Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family ( The AP2 complex also recruits synaptic vesicle proteins to endocytic sites C. elegansThe release of neurotransmitters at synapses occurs through the regulated fusion of synaptic vesicles with the plasma membrane. The recovery of synaptic vesicle membrane and proteins through endocytosis is dependent on a large complex of proteins associated with clathrin Drosophila in genetic screens for temperature-sensitive paralytic mutants Drosophila stoned mutants exhibit behavioral, electrophysiological, and ultrastructural defects that indicate that synaptic vesicle recycling is severely compromised stoned locus encodes two proteins, stoned A (STNA) and stoned B (STNB); STNA proteins are only found in insects, while proteins similar to STNB are found in all metazoans Drosophila protein, has been implicated in recycling of synaptic vesicle proteins The founding member of the stonin protein family, stoned B, was identified in Drosophila synaptotagmin Drosophila stoned mutants exhibit defects in synaptotagmin localization and synaptic vesicle recycling Drosophila rescues the lethality and synaptic vesicle recycling defects observed in stoned mutants Experiments in flies and mice suggest that stonins are linked to synaptotagmin function. STNB binds to the C2B domain of C. elegans leads to defects in synaptotagmin localization and synaptic vesicle endocytosis. The endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. No further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41/stonin acts in the same endocytic pathway as µ2 adaptin.We now show that the absence of UNC-41/stonin in C. elegans unc-41 gene result in uncoordinated movement, resistance to inhibitors of cholinesterase, slow growth, and small adult size; these phenotypes are usually associated with defects in acetylcholine (ACh) release unc-41 animals display a defecation defect associated with loss of γ-aminobutyric acid (GABA) function unc-41 encodes a protein important for the release of most or all neurotransmitter types. In addition, unc-41 mutants resemble synaptotagmin-deficient (snt-1) mutants unc-41 gene products play a role in synaptic vesicle fusion or recycling.Mutations in the unc-41 by transposon tagging, and found that it corresponds to the predicted gene C27H6.1. We note that this sequence has also been designated “apt-10” (see unc-41 locomotion and defecation phenotypes (data not shown). The longest gene product, unc-41A, consists of 12 exons with a 5′ transpliced SL1 leader ; the sizes and hybridization patterns of these transcripts are consistent with the structures of the two gene products shown in unc-41 gene transcripts.We cloned -10” see . A genom1 leader . A shortunc-41 gene encodes a homolog of Drosophila STNB and mouse stonin 2  or the unc-41B regulatory region (2343-bp upstream of exon 3) driving CFP or YFP. Expression of both reporters appeared to be restricted to the nervous system  in the ventral nerve cord . We note that expression of a single isoform (UNC-41B) from the unc-41A promoter rescues unc-41 mutant phenotypes We generated reporter constructs with the putative s system . The uncrve cord . A signiC. elegans nervous system (Drosophila and humans unc-41(e268) mutants . Anti-UNC-41 antibodies (gt216) were affinity-purified and used for immunofluorescence staining. We found that UNC-41 is specifically associated with synaptic regions in the s system . Strong s system . The dis mutants , demonstDrosophila stoned mutants, synaptotagmin is specifically reduced and mislocalized at presynaptic boutons unc-41 mutants. GFP-tagged vesicle proteins were expressed under control of their respective promoters; constructs were present as single copy insertions to avoid possible overexpression. Consistent with studies in Drosophilaunc-41 mutants, and was observed in non-synaptic regions such as commissures  were not affected by the absence of UNC-41 . Together, these results suggest that UNC-41 is required to establish or maintain the localization of SNT-1 at synapses.We extended these observations by examining the localization of GFP-tagged synaptic vesicle proteins in GABA motor neurons. As in the sublateral neurons, synaptotagmin was mislocalized along axons and was present in non-synaptic regions in proteins  as well snt-1(md290) mutants; md290 is a null allele of snt-1DrosophilaIn mammalian tissue culture cells, synaptotagmin recruits stonin 2 to the plasma membrane C. elegans, there are six synaptotagmin genes, snt-1 to snt-6. We examined the localization of GFP::UNC-41B in two different synaptotagmin triple mutants: snt-4 snt-1 snt-2 and snt-6 snt-1 snt-3, and observed that GFP::UNC-41B was localized to synapses in both of these triple mutants ; thus SNT-1 is not required for STNB localization.We expressed the synapses , and thaenotypes . We concsnt-1 unc-41 double mutants using three different allelic combinations, and we observed that the swimming behavior of these double mutants was more impaired than either of the single mutants , the tagged protein was localized to synapses  mutants  apm-2(e840) double mutant  are well-conserved, but the N-terminal one-half to three-quarters of the different proteins have very little sequence similarity.Members of the stonin family share specific structural features, but also exhibit considerable sequence diversity. The length of the protein varies among species and ranges from ∼900 amino acids in many mammals to 2099 amino acids in the trematode C. elegans UNC-41 , and Drosophila melanogaster STNB (containing seven NPF EF-hand-binding motifs but no ear-binding motifs). However, despite the apparent differences in protein interaction capabilities, we found that expression of a stonedB cDNA rescued unc-41 mutant phenotypes and transgenic STNB protein was properly trafficked to synapses  PDZ-binding motif Drosophila, all of these mutations also eliminate or affect STNA, because the two proteins are encoded by a bicistronic message The present study describes the first unambiguous knockout of a stonin in any organism. Although there are mutations that eliminate the STNB protein in unc-41 mutants have been reported previously, and include uncoordinated locomotion, reduced pharyngeal pumping, resistance to cholinesterase inhibitors, slow growth, small adult size, and elevated ACh levels e268 allele, and we have now shown that this allele confers the same (presumably null) phenotypes as the other non-transposon-associated alleles.Some phenotypes of Drosophila stoned mutants, synaptic synaptotagmin is reduced and the remaining synaptotagmin is mislocalized to axons unc-41 mutants supports a role for UNC-41 in localization of synaptotagmin to presynaptic terminals in C. elegans: counts of visible puncta in null unc-41 mutants indicates that SNT-1::GFP localization to many synapses is below the detection level of what is a ‘synapse’ to a blinded scorer  were also reduced in stoned mutants unc-41 mutants  can function redundantly. We have recently examined apa-2 mutants (deficient for α adaptin); apa-2 mutants exhibit only a small reduction in the number of synaptic vesicles. By contrast, apa-2 apm-2 double mutants exhibit a severe decrease in synaptic vesicles. These data suggest that α adaptin acts in parallel to µ2; a manuscript describing these results is in preparation.The lack of additive effects in C. elegans functions in two processes in synaptic vesicle endocytosis. It localizes synaptotagmin to synapses, possibly by recruiting synaptotagmin to sites of synaptic vesicle endocytosis. In addition, UNC-41/stonin functions with the µ2 subunit of the AP2 complex to recover membrane during endocytosis. Because synaptotagmin is required for membrane endocytosis In summary, our data suggest that UNC-41/stonin in unc-41 alleles with e allele designations were isolated at MRC, Cambridge, and were generously provided by Jonathan Hodgkin: e252, e268, and e650 were isolated by Sydney Brenner after EMS mutagenesis, e850 was isolated by Padmanabhan Babu after P32 decay mutagenesis, and e1162 and e1294 were isolated by Don Riddle after ICR191 mutagenesis. The unc-41 alleles with md allele designations were isolated in the Rand laboratory as spontaneous mutants resistant to the acetylcholinesterase inhibitor aldicarb unc-41 alleles by genetic mapping and complementation tests, and were outcrossed at least six times. The unc-41 alleles n2163 (EMS) and n2913 (diepoxybutane) were isolated by Erik Jorgensen and Bob Horvitz, and ox63 (ENU) was isolated by Erik Jorgensen. The snt-2(tm1711)III, snt-3(tm2426)V, and snt-6(tm3686)II alleles were obtained from Shohei Mitani , and the snt-4(ok503)I allele was provided by the C. elegans Gene Knockout Consortium.The wild-type strain is Bristol N2. Additional strains are in C. elegans DNA and RNA, screening cDNA and genomic libraries, and performing Northern blot analyses. The unc-41 gene was cloned by transposon tagging md1175. The genomic phage RM#231L was isolated from a C. elegans genomic library prepared by Heidi Browning and Tom Blumenthal. It contains the complete unc-41 gene with 3529 bp of upstream sequence. The genomic sequence was determined by “primer walking,” using plasmid subclones from phage RM#231L as templates. The genomic subclones were also used to screen two cDNA libraries prepared by Bob Barstead. Genomic clones and cDNAs were completely sequenced on both strands using the fmol® DNA Cycle Sequencing System . The unc-41 gene, which has the cosmid designation C27H6.1, was subsequently sequenced by the C. elegans Genome Sequencing Consortium (Genbank Accession number NM073165), with identical results. We also note that this gene was transiently known as apt-10Standard molecular biology techniques were used for preparing unc-41 genomic regions using direct single-worm PCR. The precise deletion endpoints or insertion sites were determined by sequencing purified PCR products. PCR products from those mutants without rearrangements were used for Restriction Endonuclease Fingerprinting analysis Mutations were initially analyzed by Southern blot or PCR analysis. Some of the mutations were associated with altered fragment lengths, which allowed us to determine the approximate nature of the rearrangement. Further analysis involved amplification of specific 1–2 kb unc-41 gene were cloned into the first multiple cloning site of pPD49.26. A CFP, GFP, or YFP reporter gene carrying a nuclear localization signal  was cloned into the second multiple cloning site unc-41 cDNA clone yk40f7 was obtained from Yuji Kohara , and the stonedB cDNA was generously provided by Mani Ramaswami . The GFP::UNC-41B (pMG13) construct from Jung et al.Expression plasmids for transformation utilized the pPD49.26 vector or derivatives . Promoter regions from the E. coli and used to generate polyclonal antibodies. The plasmid RM#198p contains coding sequences corresponding to residues 1124 to 1693 of the UNC-41A protein in the malE expression vector pIH902 (New England Biolabs). The resulting fusion protein was insoluble and was purified as inclusion bodies. The fusion protein was injected into two goats (gt216 and gt220). Polyclonal antibodies were affinity purified against the UNC-41 fusion protein as described previously unc-41(e268) mutants  Nematodes were stained using a modified freeze-fracture procedure as previously described C. elegans were essentially as described by Mello et al. Punc-122::GFP was injected at 50 ng/µl.DNA transformation methods for snt-1::GFP construct (pRH353) was injected at 1, 5, or 25 ng/µl into wild-type animals. The coinjection marker Punc-122::GFP was injected at 50 ng/µl. The same extrachromosomal arrays were subsequently crossed into snt-1(md290), unc-41(e268), and unc-41(md134) backgrounds. Rescue of the unc-41 phenotype was not observed in these synaptotagmin overexpression experiments. In addition, expression of a SNT-1::GFP fusion under control of the pan-neural synaptobrevin (snb-1) promoter failed to rescue the unc-41 phenotype. Because it was remotely possible that the GFP tag interfered with SNT-1 function, we also overexpressed untagged SNT-1. A snt-1 genomic plasmid (pRH264) was injected into unc-41(e268) at 25 ng/µl along with the coinjection marker Prab-3::GFP at 75 ng/µl. Rescue of unc-41 phenotypes was not observed in these transgenic animals either (data not shown).A Confocal images of UNC-41 and YFP::STNB expression patterns were collected on a Leica TCS NT confocal microscope using 40× or 63× magnification objectives, at 512×512 or 1024×1024 pixels. Images were cropped to size, assembled, and annotated using Adobe® Photoshop® CS2. All images within a given experiment were collected in the same session using identical settings and were processed identically.For all other images, worms were immobilized by using 2% phenoxy propanol and imaged on a Pascal LSM5 confocal microscope using Zeiss plan-Neofluar 10×0.3 NA or Zeiss plan-apochromat 63×1.4 NA oil objectives.Quantification of puncta in sublateral nerve cords: Young adult hermaphrodites were immobilized with 2% phenoxypropanol and imaged using a Pascal LSM5 confocal microscope with a Zeiss plan-apochromat 63×1.4 NA oil objective. Ten 100 µm regions of sublateral nerve cords (approximately midway between the level of the retrovesicular ganglion) were imaged using identical settings. The experimenter was blinded to the genotype of the animal and the identity of the fluorescent protein, and the total number of fluorescent puncta was determined for each 100 µm region.unc-41(e268), and unc-41(e268); apm-2(e840) adults were prepared in parallel for transmission electron microscopy. All animals were raised at room temperature (22.5°C). After high-pressure freezing, chemical fixation and substitution, and embedding, mutant and control blocks were blinded, and ribbons of 250 contiguous ultrathin (33 nm) serial sections were collected from two animals of each genotype using an Ultracut 6 microtome (Leica). Images were collected on a Hitachi H-7100 125keV electron microscope using a Gatan slow scan digital camera. Image analysis was performed using ImageJ software (v1.38 NIH). Axonal processes in the ventral nerve cord were reconstructed for the VA and VB ACh, and VD GABA motor neurons. Neuromuscular synapses were identified by the presence of a varicosity containing synaptic vesicles surrounding a dense projection oriented toward the muscle. The total number of neuromuscular junctions analyzed was 19 synapses (ten ACh and nine GABA) for unc-41 and 18 synapses (nine ACh and nine GABA) for unc-41 apm-2 double mutants. Vesicles were analyzed only in sections containing a dense projection. The numbers of synaptic vesicles (∼30 nm), dense-core vesicles (∼40 nm), and large vesicles (>40 nm) at each synapse were counted, and their distances from presynaptic specialization and plasma membrane were determined, as well as their diameters. The average number of synaptic vesicles and docked vesicles per profile were calculated for each set of images containing any part of the presynaptic dense projection. The numbers for each profile were averaged to obtain the final value.Wild type (N2), All behavioral measurements were performed at ∼22°C on young adult hermaphrodites. To measure swimming, individual worms were transferred to M9 buffer and allowed to adapt to the liquid environment for 60–90 s. The number of body bends was then counted for 60 or 120 s for each genotype (n ≥7). For expulsion measurements, ten young adults of each strain were observed for 10–12 consecutive defecation cycles, and the success rate was determined as the percentage of cycles that culminated in an enteric muscle contraction (EMC).t-test was used for statistical analyses.The two tail unpaired Student’s Figure S1Map of unc-41 mutations. Molecular details of each mutation are in unc-41 transcription.(TIF)Click here for additional data file.Figure S2The localization of SNT-1::GFP is disrupted in the body-sublateral nerve cords of an unc-41 mutant homozygous for the deletion allele md134 see . Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S3: Localization of synaptic vesicle proteins in GABA neurons in unc-41 mutants. Synaptic localization of SNT-1::GFP is disrupted in unc-41 mutants; fluorescence is visible in a commissure (arrowhead), although some fluorescence is also observed at puncta along the dorsal nerve cord. Minor mislocalization to axons is observed for other proteins in unc-41 mutants. Images are of dorsal nerve cords. Below: Localization of endocytosis proteins in unc-41 mutants. GFP-tagged α adaptin (APA-2::GFP) and tdTomato tagged endophilin (UNC-57::tdTomato) are not severely disrupted in the sublateral neurons of unc-41(md230) mutants. Scale bars are 10 µm.Above(TIF)Click here for additional data file.Figure S4GFP::UNC-41 is properly localized to synapses in different synaptotagmin (snt) triple mutants. GFP::UNC-41B is localized to synapses in snt-4(ok503); snt-1(md290); snt-2(tm1711) and snt-6(tm3686) snt-1(md290); snt-3(tm2426) triple mutants. Scale bar is 20 µm.(TIF)Click here for additional data file.Figure S5Dominant negative effects are observed when synaptotagmin is highly overexpressed. (A) Representative images of animals expressing a snt-1::GFP construct injected at 25 ng/µl. All images were taken at identical settings. The scale bar is 20 µm. (B) Nomarski images of a snt-1(md290) mutant, a snt-1(md290) mutant carrying a snt-1::GFP overexpressing array, an unc-41(e268) mutant, and an unc-41(e268) mutant carrying a snt-1::GFP overexpressing array. The scale bar is 100 µm. (C) Synaptotagmin overexpression does not rescue the unc-41 phenotype. The extent of rescue was quantified with swimming assays (body bends/min). Error bars indicate SEM. All t-tests are compared with the swimming rate of wild-type animals without the SNT-1 transgene. Triple asterisks denote significant difference from wild type (P<0.001).(TIF)Click here for additional data file.List S1Supplementary strain Information.(DOCX)Click here for additional data file.Table S1Molecular basis of unc-41 mutations. Alleles are listed alphabetically. Exon and amino acid numbering are for the UNC-41A protein. DNA sequences correspond to the direction of transcription. Tc1 insertions were identified at five distinct sites Click here for additional data file.


Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus.RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of  Phytoreovirus, Fijivirus and Oryzavirus [Oryzavirus. It infects plants in the family Graminae and is transmitted in a persistent manner by brown plant hoppers. The disease caused by this virus was first discovered in 1976-1977 in Indonesia and Philippines. Then the disease became prevalent in most rice-growing countries in south-eastern and far-eastern Asia, causing great yield losses to rice production [Mrs of about 71 kD, 68KD and 32KD, respectively, are non-structural proteins, whereas proteins specified by other RNA segments have been shown to be present in or are supposed to take part in the assembly of RRSV virions [Plant infecting reoviruses are grouped into three genera, namely yzavirus . RRSV beoduction . RRSV vioduction . The RRSoduction . The com virions -13. HoweTurnip crinkle virus (TCV) bind dsRNAs without size selection. These VSRs may function to protect viral dsRNAs from being cleaved by plant DCLs [RNA silencing is now a general term that refers to a set of related processes in which small RNAs ranging from 21 to 30nt in length are used to direct sequence specific modulation of gene expression . In planant DCLs ,21. Manyant DCLs . Regardlant DCLs .Oryzavirus has been reported, nor can it be predicted because of the low level of sequence similarity between proteins of reoviruses across genera. Given the importance of VSRs in virus-host interaction, we conducted experiments to identify the VSR of RRSV.The VSRs for members of the other two genera of Phytopathogenic reoviruses have been identified -26. HoweNicotiana benthamiana line 16c as described previously [Tomato aspermy cucumovirus (TAV) were used as negative and positive controls, respectively [It was reasonable to presume that the silencing suppressor function of RRSV was encoded by a non-structural protein. Therefore, the ORFs for the three non-structural proteins, pns6, pns7 and pns10, were individually cloned into the binary vector pPZP212. In leaf patches infiltrated with 35S-GFP plus 35S-S6 or plus 35S-2b, the fluorescence intensity remained strong until 7dpi  i Figure . Howeveri Figure . Similari Figure . The 35Si Figure .To test whether silencing suppression was responsible for the above observations, Northern blot analyses were conducted to detect steady-state levels of GFP mRNA and GFP-specific siRNAs. As shown in Figure N. benthamiana plant line 16c were infiltrated with Agrobacterium tumefaciens harboring 35S-ssGFP (sense GFP RNA), 35S-dsGFP (IR-GFP), and a binary vector containing S6, ΔS6, or TAV2b under the control of the 35 S promoter. As shown in Figure As mentioned above, RNA silencing is a multi-step process . To deteN. benthamiana line 16c, it could not maintain strong GFP fluorescence in infiltrated leaf patches. The GFP mRNA accumulation level was very low at 7dpi., whereas that of GFP siRNAs was markedly high  were inoculated with PVX, PVX-S6 or PVX-ΔS6, respectively.Results from the above experiments indicated that RRSV Pns6 was a silencing suppressor but offered no clues with regards to whether this function would have biological implications for viral infection. The role of Pns6 in RRSV infection can not be tested directly because of the lack of an infectious clone for this virus. Therefore, we utilized pGR107, a PVX vector to express S6 in a heterologous virus. Seedlings of N. benthamiana infected by PVX or PVX-ΔS6. However, most plants infected by PVX-S6 exhibited very severe symptoms, with some newly developed leaves being abnormal in shape (Figure N. benthamiana plants were detected. Northern blot analysis revealed that the concentration of PVX in plants infected by PVX-S6 was very high at 18dpi. In contrast, the presence of PVX RNA was hardly detectable in newly developed leaves from plants infected by PVX or PVX-ΔS6 at this time (Figure Variations in symptom severity were observed in individual infections. However, a general trend was that PVX-S6 elicited more severe symptoms than PVX or PVX-ΔS6 did. This was especially obvious after 9dpi. At this time, only mild chlorotic spots could be observed on some leaves from e Figure . The syme Figure . This ine Figure . As plane Figure , this imN. tabacum Xanthi nc and N. benthamiana plants. When transiently expressed in epidermal cells from N. benthamiana, the Pns6-eGFP fusion protein was present predominantly along the cell wall [in Trans [Taken together, we identified pns6, which is composed of 592 amino acids with a molecular weight of about 65 kDa, as a silencing suppressor of RRSV. This was the first VSR to be identified from an oryzavirus. We showed that the RNA binding activities of Pns6 might be important for its silencing suppressor function. Similar studies on other VSRs of plant infecting reoviruses have yet to be done -26. Inteell wall . This waell wall -33. For ell wall . Throughell wall . In addiin Trans . Thus, oThe authors declare that they have no competing interests.JW and ZD performed the experiments, analyzed the data, and drafted the manuscript and contributed equally to this work. CW, LC, MH and QL assisted in the performance of the experiments. ZW, YL and LX designed the study, interpreted the data and critically revised the manuscript. All authors read and approved the final manuscript.Materials and Methods. Word DOC containing the Materials and Methods section.Click here for fileTable S1: Primers used in this study.Click here for file


A total of 83 larvae of two taxonomic groups of the families Pomacentridae and Apogonidae were observed in the two water masses around One Tree Island, southern Great Barrier Reef. The study provides the first in-situ evidence that pelagic reef fish larvae discriminate reef odor and respond by changing their swimming speed and direction. It concludes that reef fish larvae smell the presence of coral reefs from several kilometers offshore and this odor is a primary component of their navigational system and activates other directional sensory cues. The two families expressed differences in their response that could be adapted to maintain a position close to the reef. In particular, damselfish larvae embedded in the odor plume detected the location of the reef crest and swam westward and parallel to shore on both sides of the island. This study underlines the critical importance of in situ Lagrangian observations to provide unique information on larval fish behavioral decisions. From an ecological perspective the central role of olfactory signals in marine population connectivity raises concerns about the effects of pollution and acidification of oceans, which can alter chemical cues and olfactory responses.The behavior of reef fish larvae, equipped with a complex toolbox of sensory apparatus, has become a central issue in understanding their transport in the ocean. In this study pelagic reef fish larvae were monitored using an unmanned open-ocean tracking device, the drifting  Despite the critical role of larval behavior in scaling population connectivity revealed theoretically by coupled biological and physical models in situ.Behavior of minute fish larvae equipped with a complex toolbox of sensory apparatus ca. 10,000 slower in water than in air (translated in hundred-fold distance disadvantage) Coastal habitats possess unique signatures To understand the chemo-sensory guidance of their homing behavior, we need to test whether and how larvae use these capabilities directly in the ocean. Studying the behavior of mm-sized fish larvae remains a major research frontier as they are too small for remote tracking One Tree Island Research Station from the University of Sydney approved the permits for this study. Permit numbers or approval ID for this study are the following: Great Barrier Reef Marine Park Authority (GBRMPA): GIO 33.239.1 and Fisheries Permit DPI 10 32 56. Larvae were caught at night in light traps and crest nets and tested the day following.The Pre-settlement stage larvae from two common reef fish families  were placed individually in a Drifting In Situ Chamber DISC,  equippedPrevious work at OTI showed that pomacentrid and apogonid larvae in flume tests can sense the difference between ocean and lagoon water preferring the lagoon odor ca. 1 mm) and is therefore open to larger scale turbulent flow and chemical cues, and transparent to sound. The DISC is linked to a surface float bearing a Global Positioning System (GIS) and to a drogue underneath that keeps it locked in the current, drifting with the water mass in which it is embedded. A subsurface float on elastic line enhances the decoupling of the underwater unit from surface waves. Its operating principle is similar to the prototype described in The observational system used in this study is an improved version of the Orientation With No-apparent Frame Of Reference (OWNFOR) experimental prototype designed to collect information about navigational cues in marine larvae Observations were carried out on both sides of OTI reef in February 2009 . The fol30) and are only indicative of relative levels of larval activity between treatments. Here we used larval positions recorded every second to compute and statistically quantify their swimming speed In enclosed circular arenas, orientation movement is typically indicated by the position of the animal corresponding to its bearing −1) were discarded Data slower than sample rate , 45 in oceanic water (‘ocean’), and 3 were in the front between the plume and the ocean and were included in the ‘plume’ treatment. Most larvae  showed significant directionality  in reef or ocean water . Those tp always >0.1, p = 0.04). However, a regression analysis did not show a significant relationship between swimming speed and temperature, both averaged per deployment (p = 0.97), Pomacentridae (p = 0.22). The activational effect of reef water on the apogonids larvae was thus unrelated to temperature.At the population level, swimming speeds were different between plume and ocean, albeit only at the 10% confidence level for Apogonidae . Indeed,ployment . The absp = 0.041) slower in the odor plume  can trigger a directional response.The concept of ‘taking the lab to the ocean’ was realized by mounting a behavioral chamber and an imaging system on a drifter equipped with an environmental sensing system. This novel system allowed us to observe differences in larval swimming speed and orientation inside and outside reef water plumes. These distinct motions could be caused by differences in chemical signature of the water, rather than temperature or current direction. Odor choice tests established that larval reef fishes can recognize reef odor and prefer it over ocean water Chromis atripectoralis) by observers equipped with scuba found a South-Southeast larval orientation on the leeward and windward sides of Lizard Island during the day ca. 1 km from the reef crest , in conjunction with propagation experiments of the reef soundscape It is important to note that both odor choice studies and ours concluded that temperature, normally a strong signal in the ocean, did not appear to influence the observed behavior . Thus odef crest . Tacklinca. 20 cm.s−1) ca. 10 cm.s−1 across the arena  from the open ocean. This requires both a sense of direction (compass) and geographic location (map) or/and access to familiar cues. Discrete reef odor sources could be determined by associating particular odors with the ebb flow coming from different locations of One Tree Island, or from other islands of the Capricorn Bunker Reef in situ Lagrangian observations.Our new information would add reef odor to larval transport and recruitment models in situ will be necessary to elucidate the sensory processing sequence of pre-settlement fish larvae; for example, introducing reef odor into the behavioral arena set adrift in blue waters, outside the tidal halo, will help isolate the larva’s response from that to reef sounds. Importantly, the central role of chemo-sensory information and odor maps in the early life history of fish raises concerns of compounded effects of habitat fragmentation, ocean acidification and chemical pollution altering olfactory discrimination In sum, we propose that (home) reef odor acts as a wake up call, or a signal informing the animal to modify its swimming speed and to switch to other sensory information for direction toward the reef. Both types of responses to reef-born odor plume, i.e., reducing swimming speed and sensory switching by orienting alongshore or mapping olfactory signal by infotaxis, would increase the chances of successful settlement. We can only speculate about the nature of other sensory signals  as the sequence of settlement cues needs further observations. Cue manipulation Figure S1Movement analysis of individual fish larvae deployed in the Drifting In Situ Chamber (DISC) at One Tree Island (OTI) on the Great Barrier Reef, February of 2009. A) cardinalfish larva of the species Cheilodipterus quinquelineatus (Family: Apogonidae) in plume water (deployment #55); B) cardinalfish larva C. quinquelineatus in ocean water (deployment #38); C) damselfish larva of the species Pomancentrus moluccensis (Family: Pomacentridae) in plume water (deployment #1); D) damselfish larva P. coelestus in ocean water (deployment #16). Subplots represent: a) density distribution of the fish larva’s swimming speeds; b) fish larva’s original trajectory in the chamber’s frame of reference; c) fish larva’s trajectory in the cardinal reference, i.e., corrected by the compass rotation. The larva’s movement is sampled every second for a total of 15 minutes (or 900 s); the larva’s trajectory is color-coded by time in seconds. The x-axes on subplots (a) have different scales since damselfish are faster swimmers than cardinalfish.(PDF)Click here for additional data file.Text S1Statistical Analyses.(DOC)Click here for additional data file.


Escherichia coli, Neisseria gonorrhoeae, Salmonella enterica, Streptococcus pyogenes and Xenorhabdus nematophila. We find that the transcriptome maps generated by our algorithms are highly accurate when compared with focused experimental data from E. coli and N. gonorrhoeae, and we validate our system’s ability to identify novel small RNAs, operons and transcription start sites. Our results suggest that Rockhopper can be used for efficient and accurate analysis of bacterial RNA-seq data, and that it can aid with elucidation of bacterial transcriptomes.Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of bacterial transcriptomes. However, computational methods for analysis of bacterial transcriptome data have not kept pace with the large and growing data sets generated by RNA-seq technology. Here, we present new algorithms, specific to bacterial gene structures and transcriptomes, for analysis of RNA-seq data. The algorithms are implemented in an open source software system called Rockhopper that supports various stages of bacterial RNA-seq data analysis, including aligning sequencing reads to a genome, constructing transcriptome maps, quantifying transcript abundance, testing for differential gene expression, determining operon structures and visualizing results. We demonstrate the performance of Rockhopper using 2.1 billion sequenced reads from 75 RNA-seq experiments conducted with  Transcriptome assays are increasingly being performed by high-throughput RNA sequencing (RNA-seq) methods. As compared with microarrays, high-throughput sequencing technologies have a number of advantages, including single base pair resolution, low background signal, a large dynamic range of expression levels over which transcripts can be detected, higher levels of reproducibility, smaller sample requirements for starting RNA and no limitation in detecting transcripts that do not correspond to a previously sequenced genome . High-thOne of the challenges associated with RNA-seq experiments is analysis of the large resulting data sets. There are numerous tools available [reviewed in ] that suEscherichia coli and Neisseria gonorrhoeae. Rockhopper is available at http://cs.wellesley.edu/∼btjaden/Rockhopper.We present a system and methods designed specifically for analysis of bacterial RNA-seq data. Our system includes new algorithms, which integrate models for sense and antisense sRNAs as well as operon structures, for constructing bacterial transcriptome maps based on RNA-seq data. Starting with sequencing reads output by high-throughput sequencing technology from one or more RNA-seq experiments, our system, called Rockhopper, aligns the reads to a genome, normalizes data from different sequencing experiments, assembles transcripts and identifies transcript boundaries including UTRs and novel small RNAs, quantifies transcript abundance, tests for differential gene expression between experiments, identifies operon structures and enables visualization of the results in a genome browser. The Rockhopper workflow is depicted in N.gonorrhoeae, strain N. gonorrhoeae F62 was used in this study  according to the manufacturer’s instructions to remove contaminating DNA. RNA was then ribosomal RNA (rRNA) depleted using the MicrobeEXPRESS kit (Ambion) according to manufacturer’s instructions. A complementary DNA (cDNA) library of the resulting messenger RNA (mRNA) was then prepared using BioChain’s Directional mRNA Sample Prep kit according to the manufacturer’s instructions. Briefly, RNA was fragmented with metal ion scission and treated with PNK. Following PNK treatment, RNA was purified, and 5′ and 3′ Cap and Tail adapters were added. Reverse transcription and 15 cycles of PCR using primers analogous to the adapters generated a cDNA library. This library was then sequenced using 36 or 40 base pair reads on Illumina’s GAIIX machine. As the genome sequence of strain F62 is not available, the sequencing reads were aligned to the genome sequence of N.gonorrhoeae FA1090 (RNA was isolated from e FA1090 .Escherichia coli strains and plasmids used in this study are listed in Supplementary Table S1. Escherichia coli strain MG1655 was grown in LB medium to OD600∼0.1 at 37°C and exposed to 0.5% α-methylglucoside (αMG) for 15 min. Escherichia coli strain CV104 harboring either pHDB3 or pLCV1 plasmids was grown in morpholinepropanesulfonic acid (MOPS) minimal medium supplemented with 0.2% D-glucose to OD600∼0.5 and exposed to 0.1 mM IPTG. Total RNA was extracted using the hot phenol method as described previously  wild-type or a mutant Salmonella strain carrying a deletion of the sRNA, sgrS, (DB111) was grown in minimal MOPS medium (Teknova) to mid-logarithmic phase. Total RNA was extracted using hot phenol , which models RNA-seq experiments in silico. First, using the Flux Simulator, we simulated an artificial expression profile based on the 2002 annotated genes in the N. gonorrhoeae genome  were designed to be 100 bp downstream of transcriptional start sites as determined by RNA-seq and were labeled using T4 PNK for 60 min at 37°C followed by 2 min at 90°C to inactivate the PNK. Following probe hybridization to bacterial RNA, the probes were extended using reverse transcriptase for 1 h at 41°C. Single-stranded DNA products were then run through an 8% TBE–Urea gel along with a single-stranded DNA ladder, which was also radiolabeled. Size of primer extension products was determined by plotting the base 10 logarithm of the size of each ladder marker against distance traveled in the gel on semi-log paper. Primer extension products were then sized using the resulting standard curve.For primer extension, 10 μg of bacterial RNA was incubated with a . Most cSupplementary Table S7. In summary, among the tools, the percentage of the ∼2.1 billion reads that were successfully aligned ranged from 69 to 78%, and the execution time per million reads per processor ranged from 51 to 135 s  values are used as gene expression measures . Howeveret al. .Bacterial genome annotations typically indicate translation boundaries (start and stop sites) for most protein coding genes and transcription boundaries for many RNA genes, primarily rRNAs and transfer RNAs. However, annotations typically lack transcription boundaries for protein-coding genes, and they lack annotations for many small non-coding RNA genes. Here, we present a novel approach for constructing a bacterial transcriptome map consisting of the location of every transcript evinced by a set of RNA-seq experiments. The transcriptome map is determined, first, by identifying a set of transcript seeds consisting of annotated genes and high confidence novel transcripts corresponding to genomic regions where a significant number of reads were found to align. The transcript seeds are then extended using a Bayesian approach to identify more precise transcript boundaries . Transcriptome maps were generated from RNA-seq data for five different bacteria , the length of the 5′ UTR identified by Rockhopper is within 7 nt of the length of the 5′ UTR as determined from 5′ RACE. For one of the seven genes (cspE), our method overestimates the length of the 5′ UTR, as compared with the 5′ RACE results, by 25 nt. For one of the seven genes (panD), our method underestimates the length of the 5′ UTR, as compared with the 5′ RACE results, by 35 nt.As further evaluation of our approach for identifying transcript boundaries, we performed 5′ RACE on seven randomly chosen genes in N. gonorrhoeae as determined by primer extension and 5 of 7 genes in E. coli as determined by 5′ RACE). For the remaining cases, Rockhopper mis-identified the transcription start site by between 15 and 89 nt. There was no significant correlation between the accuracy of Rockhopper’s transcription start site identifications and gene expression levels or 5′ UTR lengths. We did not evaluate Rockhopper’s ability to identify transcription start sites when using differential RNA-seq (dRNA-seq) data based on 5′ end selection of primary transcripts, which enables more reliable discernment of precise transcription start sites  to the experimentally evinced transcription start site 59% of the time . The simulated RNA-seq experiment generated 31 million reads of length 72 nt based on expression from the annotated genes in N.gonorrhoeae using an average transcription start site deviation of 25 nt . The advantage of using a simulated RNA-seq experiment, as opposed to a non-simulated RNA-seq experiment, is that the location and abundance of every transcript is known a priori; therefore, the transcriptome map generated by our method can be compared precisely to the set of true (simulated) transcripts. Any novel transcript identifications by our method must be false positives, as all transcripts are known in the simulated experiment. Based on the simulated experiment, Rockhopper generated a transcriptome map consisting of a single novel transcript identification. This lone false positive transcript identification suggests an upper bound on the specificity of our transcriptome map.To estimate a lower bound on false positive novel transcript predictions, we evaluated our method on data from a N. gonorrhoeae with gene expression levels determined from qRT-PCR experiments. qRT-PCR was performed for nine N.gonorrhoeae genes from three biological replicates. N. gonorrhoeae RNA-seq data are used, the RNA-seq estimated expression levels have a correlation of 0.55 with the qRT-PCR data. We then estimated gene expression levels using different sized subsets of randomly chosen sequencing reads and found that the correlation with qRT-PCR results decreased as smaller subsets of reads were used, but only gradually, as the number of aligned reads decreased from 15 to 2 million.Common measures for quantifying gene expression from RNA-seq experiments are (RPKM)  and FPKMN. gonorrhoeae, we evaluated the correlation between Rockhopper’s estimated transcript abundance levels and the known simulated expression levels for all 2002 annotated protein-coding genes. When all simulated reads were used, the estimated gene expression levels had a correlation of 0.96 with the known simulated expression levels  between GC contents and expression levels of transcripts based on our RNA-seq data. We did not explore more complex relationships, beyond correlation, between GC-content and expression, though it may well be the case that GC content biases are unimodal, with both high GC content and low GC content transcripts being under-represented by sequencing reads , the variability between the ‘surrogate’ replicates is expected to be larger than the variability between ‘true’ replicates would be, and weaker conclusions are drawn. Rockhopper uses information from biological replicates when available. When biological replicates are not available, Rockhopper uses surrogate replicates.Supplementary Figure S3). Local regression, Lowess, is used as in Supplementary Figure S3 to obtain a smooth estimate of the variance . Because multiple tests are being performed across the set of genes, q-values are reported that control the false discovery rate using the Benjamini–Hochberg procedure  estimated to be upregulated on αMG addition based on the RNA-seq data were corroborated by the RT-qPCR data. One of the three genes (ydjN) estimated to be downregulated by αMG addition based on the RNA-seq data was corroborated by the RT-qPCR data. Though the direction of regulation determined from the RNA-seq data was confirmed by RT-qPCR for 7 of the 10 genes, the fold change determined from the RNA-seq data was generally underestimated. For 3 of the 10 genes , the direction of regulation determined from the RNA-seq data disagreed with that determined by RT-qPCR.To evaluate Rockhopper’s ability to identify differentially expressed genes based on RNA-seq data, we used RT-qPCR to investigate 10 randomly chosen rbsD and ibpA, all alignment tools used in this study  mapped fewer RNA-seq reads to the gene in the +αMG condition than in the −αMG condition, even though the RT-qPCR data suggest that these two genes are upregulated in +αMG relative to −αMG. Similarly, for sthA, all alignment tools used in this study mapped more RNA-seq reads to the gene in the +αMG condition than in the −αMG condition, even though the RT-qPCR data suggest that this gene is downregulated in +αMG relative to −αMG. As evinced by the error bars in For the three genes where the direction of regulation determined from RNA-seq data disagreed with the direction evinced by RT-qPCR, we investigated whether the disagreement was likely a result of the computational analysis or of the underlying RNA-seq data. For Multi-gene operons are a means by which bacteria can implement co-expression of related genes, and systematic identification of operons is an important step toward reconstruction of regulatory networks. Numerous computational approaches have been proposed, based on various features suggestive of polycistronic messages, for predicting operon structures throughout a genome [reviewed in ]. When eWe investigated the extent to which operon structures can be predicted based on RNA-seq data. We used two features to estimate the probability that consecutive genes on the same strand are co-transcribed: the distance in nucleotides between the genes and the similarity of the genes’ expression in the RNA-seq data. The features are combined, together with a prior probability, using a naïve Bayes classifier to determine the probability of co-transcription .E. coli as reported in RegulonDB  contained different numbers of genes, ranging from 2 to 11. For the 10 sets of genes, we used RT-PCR to test for co-transcription of 13 pairs of genes that were reported to be co-transcribed in RegulonDB and of 14 pairs of genes that were not reported to be co-transcribed in RegulonDB . As data from each RNA-seq experiment are normalized, results from different experiments can be viewed simultaneously and meaningfully compared.Results from Rockhopper are displayed using the Integrative Genomics Viewer  to enablbacterial RNA-seq data analysis. Although tools for analysis of eukaryotic RNA-seq data are often applied to bacterial RNA-seq data, the tools are limited by their lack of appropriate modeling of bacterial gene structures and transcriptomes, including operon structures, sRNAs and dense genomes with overlapping genes.RNA-seq experiments have proven to be a powerful approach for assaying bacterial transcriptomes. However, analysis of the large resulting data sets can be a limiting factor for studies using RNA-seq experiments. There are numerous tools available that support various aspects of RNA-seq data processing, management and analysis, but typically for a single study using RNA-seq experiments, a variety of tools must be used, often in a piecemeal fashion and possibly requiring the user to possess computational expertise. Importantly, there is a paucity of tools designed specifically for Here, we present a set of algorithms for analysis of bacterial RNA-seq data, implemented in a user-friendly open-source software tool named Rockhopper. We evaluated Rockhopper on >2 billion reads from 75 RNA-seq experiments conducted using five different bacteria. Our motivation for using a range of RNA-seq experiments is to help us understand the robustness of our methods when applied to RNA-seq data from experiments using different bacteria under disparate experimental conditions. When aligning reads to a bacterial genome, we found that Rockhopper had comparable or higher accuracy than the four leading tools that we evaluated.N. gonorrhoeae included 34 small transcripts that we identified as candidate sRNAs. Eleven of these candidate sRNAs were evaluated in another study by comparison to the Rfam database , yielded reads with a lower average quality score (31 as compared with 35) and resulted in fewer reads on average being aligned to the genome (55% as compared with 80%). In E. coli, for 7 of 10 genes examined, Rockhopper’s differential expression estimates agreed with those from RT-qPCR data. For the 3 of 10 genes that disagreed, our investigations suggest that the disagreement stems not from the statistical test for differential expression but from a discrepancy between the RNA-seq data and the RT-qPCR data. Interestingly, when we used randomly chosen subsets of reads from our N. gonorrhoeae RNA-seq experiments, above about a million reads of length 36 nt each for a 2.2 megabase genome, we did not find correlation between qRT-PCR expression levels and those estimated by Rockhopper to change significantly. These results provide a small window of insight into the depth of RNA-seq coverage necessary so as not to compromise on the accuracy of gene expression estimates. Indeed, on an Illumina HiSeq 2000 system, we observed no degradation in read quality or in downstream analyses of read data when multiplexing up to eight samples per lane, resulting in >200 million reads per lane and ∼27 million reads per sample. We did not try multiplexing more than eight samples per lane. These findings suggest that when cost of sequencing is a constraint, multiplexing large numbers of samples may be an option, though it results in fewer reads per sample, it may not significantly impact the accuracy or reproducibility of analyses of the data.Rockhopper also estimates transcript abundances and tests for differential gene expression between experimental conditions. We identified 1827 genes in E.coli RNA-seq data from 16 conditions, was able to characterize 90% of experimentally confirmed operons in E.coli, and we confirmed via RT-PCR six previously uncharacterized operons that were identified by our approach using RNA-seq data from N.gonorrhoeae and 12 previously uncharacterized operons that were identified by our approach using RNA-seq data from E.coli. We did not investigate Rockhopper’s ability to discern operons controlled by multiple promoters, though we expect instances of such operons to be increasingly revealed as the use of bacterial RNA-seq experiments grows.Operons are a means by which multiple genes can be controlled by a single regulatory system in bacteria. Understanding which sets of genes are co-transcribed can help illuminate regulatory networks. We describe a novel approach for characterizing bacterial operon structures based on RNA-seq data. We demonstrated that this approach, using Computational methods for analysis of bacterial RNA-seq data need to keep pace with the increasing use of RNA-seq experiments that assay bacterial transcriptomes. The large data sets that result from RNA-seq experiments necessitate systematic analyses that are both accurate and reproducible. The Rockhopper system is one attempt at such a set of systematic analyses for bacterial RNA-seq data. Rockhopper is integrated with a genome browser to enable exploration of RNA-seq data and visualization of analysis results. Rockhopper is designed as a first step following an RNA-seq experiment so as to facilitate investigation of the myriad downstream questions that bacterial researchers may wish to explore.Supplementary Data are available at NAR Online: Supplementary Tables 1–8, Supplementary Figures 1–4 and Supplementary References .National Science Foundation (NSF) [MCB0919808 to B.T.] and the National Institutes of Health [R01AI048611 to C.G. and R.M.]. Funding for open access charge: NSF grant [MCB0919808 to B.T.].Conflict of interest statement. None declared.


The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays.TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability.We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between  Functional roles for both coding and non-coding RNA molecules have been increasingly appreciated in a variety of biologic processes, including gene regulation, molecular trafficking, and protein translation -4. For eHRAS RNA and CNBP protein, as well as human Stau1 and TP53 RNA and show that Stau1 influences TP53 RNA stability in the context of transcriptional blockade.The recent use of protein microarrays in yeast ,18 suggeTP53, HRAS, MYC, BCL2 and non-coding RNAs PWRN1, SOX2OT, OCC1, IGF2RNC, lncRBM26 and DLEU1. The schematic diagram of the workflow used in this work is presented on Figure To define RNA-protein interactions, we utilized sense and antisense strands for 10 RNA transcripts representing protein coding RNAs HRAS sense RNA incubation to the microarray. Comparison of the ratio of signal intensity above background of every spotted protein between the two independent replicates demonstrated suitable results with a Pearson correlation of r2=0.869  is shown on Figure HRAS sense RNA probed to protein array resulted in a significance of p < 10-72  for further domain analysis Table . InteresRNA application to human protein microarrays therefore identified a set of human proteins with a broad capacity for binding to multiple RNAs that contain known RNA binding motifs as well as identified a number of new proteins for future study that contain no canonical motifs and no previously known RNA binding capacity. To further narrow the lists of candidate RNA-protein interactions, we selected proteins that significantly bound sense, but not antisense RNAs, although some biologically relevant interactions may take place with both sense and antisense transcripts from a given locus. This analysis resulted in 137 interactions, revealing a substantial range of protein binding between individual mRNAs and non-coding RNAs and identified specific interactions for further study Figure .TP53 mRNA using protein microarrays with signal over background 3.19-fold, Z-score 4.24  was identified as a protein-binding target of  Figure . TherefoHRAS mRNA on the protein microarray with signal over background 3.62-fold, Z-score 3.82 , which bound TP53 association with Stau1 protein, several deletion mutants spanning the UTRs of TP53 RNA were generated and used for Stau1-HA protein pull-down  in TP53 negative H1299 cells, STAU1 knock down caused reduction in the full-length TP53 RNA half-life but did not affect TP53-ORF RNA after actinomycin D treatment  , RNA tra Figure . Moreove Figure . These iThe diversity of naturally occurring RNA-protein interactions is only beginning to be appreciated and RNA hybridization to human protein microarrays may be a useful complement to current platforms in a number of ways. For example, this approach can be used to gain mechanistic insight into newly identified long non-coding RNAs by understanding the proteins to which they bind. The ability to test the binding capability of any coding or non-coding RNA to thousands of proteins simultaneously will significantly improve the pace of mechanistic analyses of RNAs. In this regard, it may provide a complement to biotin-labeled RNA pull-down/mass spectrometry-based approaches, which have proven successful in identifying RNA binding proteins  but requTP53 in an as yet previously uncharacterized way. Similarly, the binding of CNBP to HRAS RNA may also modulate Ras function. Moreover, it is intriguing to note that human genetic disorders arising from mutations in both CNBP (Myotonic Dystrophy  was recloned from the original pSPORT1-TP53 vector as a SalI-ClaI fragment of 2560 bp into SalI-EcoRV of pSPARTA. The TP53 deletion mutants containing 5′UTR and 197bp of 3′UTR were constructed by flanking TP53 fragments with either HindIII-XhoI or EcoRI-XhoI and cloning into pcDNA3 expression vector to generate pcDNA3-TP53-ORF-5′UTR and pcDNA3-TP53-ORF-3′UTR197. The constructs containing TP53-ORF, TP53-ORF-3′UTR (full-length) and TP53-ORF-3′UTR453 fragments were generated by direct amplification and TA cloning into pcDNA3.1TOPO expression vector. Numbering for 3′UTR deletion constructs corresponds to the first nucleotide following the termination codon, defined as 1. The full-length TP53 expression construct used for transfection of H1299 cells was constructed by recloning HindIII-NotI fragment from pSTARTA-TP53 into pcDNA3 in order to take advantage of the neomycin selection cassette in H1299 cells.Plasmid vectors containing full-length transcribed sequences for coding and non-coding RNAs were obtained from Open Biosystems (Thermo Scientific) and are described in detail in Additional file ′ end for Stau1 and 5′ end for CNBP using PCR techniques and the following primers:Human V-Ha-Ras homolog [GenBank:BC006499] was recloned from pOTB7 vector as 1146 bp EcoRI-XhoI fragment into EcoRV-XhoI of pSPARTA. Both plasmids pSPARTA-TP53 and pSPARTA-HRAS were used in pull-down experiments in vivo as described below. In vivo expression constructs for Stau1 and CNBP were generated using pcDNA3.1Hygro plasmid (Invitrogen). The ORFs of Stau1 and CNBP were flanked with 3x hemagglutinin (HA) tag at 3′TTTTAAGCTTACCATGTCTCAAGTTCAAGTGCAAGTT.STAUF, 5′TTTTCTCGAGTCAGGCGTAATCGGGCACGTCGTASTAUHAR, 5GGGATAGCTTCCTGCATAATCAGGGACGTCATAGGGATAGCCAGCATAGTCAGGCACATCGTATGGGTAGCACCTCCCACACACAGACA.′TTTTAAGCTTACCATGTACCCATACGACNBPHAF, 5TGTGCCTGACTATGCTGGCTATCCCTATGACGTCCCTGATTATGCAGGAAGCTATCCCTACGACGTGCCCGATTACGCCAGCAGCAATGAGTGCTTCAAG.′TTTTCTCGAGTTAGGCTGTAGCCTCAATTGTG.CNBPR, 5The epitope tagged fragments were cloned in HindIII-XhoI sites of pcDNA3.1Hygro and the final constructs pcDNA3.1Hygro-STAU1-HA and pcDNA3.1Hygro-HA-CNBP were used for RNA pull-down studies.TP53, MYC, HRAS, BCL2) and non-coding  genes were in vitro transcribed in both sense and antisense directions using T7, T3 or SP6 promoters . We first carefully optimized the labeling procedure in order to achieve between 1 to 3 Cy5 dyes covalently attached to RNAs used. This was accomplished via the following modifications from the original manufacturers protocol: the total reaction volume was kept at 25 μl, the ratio of RNA:Label IT Cy5 reagent at 10:1 (w:v) and reaction time not more then 30 minutes at 37°C. Briefly, 5 μg RNA in water was mixed with 5 μl Label IT Cy5, diluted 1 to 10 in water to obtain a final volume of 25 μl and incubated 30 minutes at 37°C. The reaction was stopped by addition 2.5 μl of 10x STOP buffer (Mirus). The volume was increased to 100 μl with water, supplemented with glycogen (Invitrogen) to final concentration of 0.2 μg/μl, mixed and RNA ethanol precipitated in the presence of 0.5 M NaCl at −20°C for at least 1 hour. The labeled RNA was extensively washed in 70% ethanol, dried and resuspended in 16 μl water. RNA labeling density was evaluated using NanoDrop 1000 spectrophotometer (Thermo Scientific) and visualized using denaturing agarose gel-electrophoreses.RNA labeling for microarray incubations was performed using The efficacy of Cy5 dye incorporation was calculated as dye density (pmol Dye: μg RNA) and RNA Base:Dye ratio. To calculate the dye density following formula was used:dye/edye(pmol)/MRNA, (μg), wherepmol Dye : μg RNA = Adye – Cy5 absorbance at Imax (excitation wavelength) 649 nm,Adye – Cy5 dye extinction coefficient 250 000 M-1 cm-1,eRNA – RNA amount (μg).MBase:Dye ration was calculated using following formula:basex edye)/(Adyex ebase), where(Abase = A260 – (Adye x C.F.260) – RNA base absorbance,A260 – absorbance of the nucleic acid, C.F.260 – correction factor for Cy5=0.05,Abase – RNA extinction coefficient 8250 M-1 cm-1eThe RNA labeling density and Base/Dye labeling ratio for each 20 sense and antisense RNAs used in this work is presented in Additional file RNA labeling with Biotin-16-UTP was performed during in vitro transcription. First, plasmid DNA containing transcribed sequence was digested with enzymes immediately flanking the insert and 4 μg of linear DNA was used for RNA biotinilation reaction in 50 μl total volume consist of: 1x Transcription buffer (Promega), 1 mM ATP, 1mM CTP, 1mM GTP, 0.95 mM UTP, 0.05 mM Biotin-16-UTP (Roche), 10 mM DTT (Promega), 40 units RNAseOUT (Invitrogen) and 60 units RNA polymerases . The reaction was carried out at 37°C for 4 hours after which DNA was digested by addition of 2 units DNAse I at 37°C for 15 minutes. Next, RNA was phenol chloroform extracted, and after ethanol precipitation, characterized using NanoDrop 1000 spectrophotometer (Thermo Scientific) and visualized using denaturing agarose gel-electrophoreses  with custom modifications including a modified bottom gasket and top spacers , 1% BSA (w/v) , 100 μg/ml Yeast tRNA, 20 μg/mL heparin and 1 mM DTT for 1 hour at room T°C with gentle agitation. After completion of the blocking step, 10 pmol Cy5 labeled RNA was added to 0.7 mL binding buffer BB: 40 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 0.5 mM magnesium acetate, 10 μg/ml Yeast tRNA, 10 μg/mL heparin, 1 mM DTT, 0.01% Igepal CA-630, 5% glycerol, 0.2 units/μl RNAseOUT. The blocking buffer was replaced with binding buffer containing labeled RNA and microarray slides incubated in the dark at 25°C for 1 hour with gentle agitation. Following RNA incubation binding buffer was removed and washing steps were implemented using 3 times exchange of 0.75 mL WB buffer: 40 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 0.5 mM magnesium acetate, 10 μg/ml Yeast tRNA, 10 μg/mL heparin, 1 mM DTT, 0.01% Igipal 40, 5% Glycerol, 0.2 units/μl RNAseOUT for at least 5 minutes each. Finally, the microarray slide was washed in 0.75 mL washing buffer WBF: 40 mM Tris–HCl (pH 8.0), 150 mM sodium chloride, 0.5 mM Magnesium acetate, 10 μg/ml Yeast tRNA, 10 μg/mL heparin, 1 mM DTT, at least 3 times for 5 minutes each. After the last wash, the WBF buffer was removed partially in order to prevent the slide from drying prematurely and slide holder device disassembled immediately. The probed microarray slide was placed in ArrayIt microarray high-speed centrifuge and the residual WBF buffer removed via a 30 seconds of centrifugation. The dry slide was scanned at 635 nm (Cy5) using a GenePix 4000B Microarray scanner (Molecular Devices) immediately after or at least within 2 hours of the completion of the incubation. All raw and processed data is publicly available at the Gene Expression Omnibus under accession GSE34794 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ttehdwscqcgckrg&acc=GSE34794). For the visualization process, the array images from antisense RNA incubations were pseudocolored green and overlaid with the sense RNA incubation. The intensity of the 635 nm wavelength signal at each spotted protein location was determined with GenePix Pro 6.1 software (Molecular Devices). To quantify RNA-protein interactions, the intensity of 635 nm signal (F635) was divided by the local background intensity (B635) at each of the duplicate spots for a given protein. Data was filtered based on signal above the background for each of the duplicate feature to be greater than 2.5 fold and Z-Score ≥ 3 from the global mean signal from all of the spotted proteins. In order to select sense-specific protein interactions, the signal generated from antisense RNA incubations were calculated based on 2.5 fold above the background and Z-Score ≥ 3. Significant hits from the antisense were subtracted from the significant hits in the sense RNA. Hierarchical clustering analysis was performed by Cluster 3.0 (Eisen Lab) and visualized with TreeView. Gene Ontology analysis and PFAM domain analysis of RNA binding proteins was performed with DAVID . The quantification of the incubation results shown on the right. (F) Contrary to STAU1 depletion, UPF1 KD has no affect on TP53 RNA decay in Primary Fibroblasts.Click here for file


The correct full authors' addresses should be as follows:Department of Colorectal Surgery, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, 270 Dong An Road, Shanghai 200032, China.


D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5′ phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5′ di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5′ end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K Flaviviridae family (genus Flavivirus), which are small RNA viruses of 10–11 Kb in length with capped non-polyadenylated positive strand genomes. Dengue virus proteins are produced from a single open reading frame via translation of the genomic viral RNA as a single polyprotein that is co-translationally processed into 3 structural proteins  and 8 non-structural proteins . The non-structural proteins are responsible for directing viral genomic RNA replication, including synthesizing negative- and positive-strand RNAs and forming the viral RNA cap structure.Dengue viruses are members of the 7-GpppA-me2) generated by the virus encoded RTPase (NS3), GTase (NS5), 2′-OMTase (NS5), and Guanine-N7-MTase (NS5) The flavivirus RNA cap is critical for viral polyprotein translation and RNA replication. The RNA cap allows the viral RNA to be efficiently translated by the cellular translational machinery and provides protection for the genome from cellular exonucleases. Flavivirus genomic RNA replication occurs on rough endoplasmic reticulum membranes in membranous compartments away from the cellular capping machinery, requiring the viruses to develop a mechanism for generating an RNA cap structure. Dengue and other flaviviruses have evolved a complete RNA capping machinery to form an RNA cap on the 5′ end of the positive-strand genomic RNA. Cellular RNA cap structures are formed via the action of an RNA triphosphatase (RTPase), guanylyltransferase (GTase), N7-methyltransferase (N7-MTase), and 2′-O methyltransferase (2′O-MTase) in silico molecular dynamics docking of an RNA into the crystal structure of the dengue capping enzyme During the GTase reaction, the NS5 N-terminal capping enzyme must bind to the 5′ end of the viral RNA. The GTase reaction uses two substrates, a covalently bound guanosine monophosphate (GMP) and the diphosphorylated 5′ end of the viral genomic RNA, to form the cap 0 structure (5′ GpppAGUAA…). We and others have studied how the capping enzyme binds GTP In this manuscript we examine the binding of the viral 5′ diphosphorylated RNA substrate to the dengue virus capping enzyme. We developed a fluorescence polarization-based RNA binding assay to monitor the association of a short diphosphorylated RNA corresponding to the conserved 5′ end of the flavivirus genome and determined the RNA binding affinity to the capping enzyme. We assessed the effects of the various ligands used by the capping enzyme on RNA binding affinity, and determined that binding is negatively affected by GTP and ATP and positively affected by SAM. We also performed a structure-directed mutational analysis of the dengue 2 capping enzyme to determine which amino acids may be involved with RNA binding based on the structural similarity of the dengue virus capping enzyme with the Vaccinia virus VP39 methyltransferase protein bound to RNA. We identified several residues that are critical for binding to RNA and report their relative contribution to binding. We have also explored the contribution of the 5′ phosphates to RNA binding and found that the 5′ β- and α- phosphates are critical for diphosphorylated RNA binding to the capping enzyme.E. coli cells (Novagen). Cultures (750 ml) were induced with 400 µM IPTG overnight at 22°C, and the bacterial pellets were collected and stored at −80°C in low imidizole lysis buffer. Frozen pellets were thawed and lysed with a M-110-L Pneumatic microfluidizer (Microfluidics Inc.), and the lysate was clarified by centrifugation at 18 K RPM in a SS-24 rotor and filtered through a 0.22 µM syringe filter. The histidine-tagged protein was purified from clarified lysates using a Hi-Trap Nickel column  on an AKTA Purifier FPLC system. The eluted proteins were concentrated using 10 K Amicon Ultra concentrators (Millipore), and buffer exchanged into 400 mM NaCl, 20 mM Tris-Base pH 7.5, 0.02% sodium azide, 20% glycerol, and 5 mM Tris(2-Carboxyethyl) phosphine hydrochloride (TCEP-HCl) on a Superdex 200 gel filtration column (Amersham). Purified proteins were concentrated using 10 K Amicon Ultra concentrators to 100 µM and the concentrations were determined by the absorbance at 280 nm on a NanoDrop 2000 spectrophotometer  using extinction coefficients obtained from the ExPASy web site. Isolated proteins were >98% pure as estimated from SDS-PAGE and Coomassie Blue staining.Recombinant dengue virus type 2, yellow fever virus, and West Nile virus capping enzymes were previously described D values for RNA binding were determined using Kalidagraph (Synergy Software) using an equation based on D. ppAGUAA-FAM and pAGUAA-FAM had average free and bound mP values of 125 mP and 325 mP, respectively, whereas AGUAA-FAM had average free and bound values of 155 mP and 425 mP.Fluorescence polarization (FP) RNA binding assays were performed with purified wild-type and mutant dengue 2 capping enzymes and 3′-fluoroscein (FAM) labeled 5-base RNAs . The RNAs were chemically synthesized , HPLC purified, and verified by mass spectrometry. SAM and SAH were purchased from New England Biolabs. GTP, ATP, GDP, and GMP were purchased from Sigma-Aldrich. All FP experiments were performed in 50 µl volumes in black 384-well microtiter plates. Binding reactions were carried out in final concentrations of 50 mM Tris pH 7.5, 0.1% NP-40, 2 mM DTT, 50 mM NaCl, and 50 nM RNA. Wild-type and mutant dengue capping enzymes were serially diluted in 400 mM NaCl, 50 mM Tris pH 7.5, and 2 mM DTT and added to the RNA mix. The binding reactions were incubated at 28°C for 1 hr, then FP and total fluorescence signals were detected using a Victor 3 V multimode platereader set to 28°C (Perkin Elmer). All FP experiments were performed in triplicate with free RNA and total bound RNA controls. Khttp://topmatch.services.came.sbg.ac.at) Structural alignment of the dengue virus capping enzyme (PDB code: 2P1D) with the Vaccinia VP39 methyltransferase domain (PDB code: 1AV6) was performed with the TopMatch Alignment Server ” sequence, and we observed that a 5′ ppAG terminated RNA not related to a flavivirus 5′ UTR can be capped by the capping enzyme D for ppAGUAA RNA binding to dengue 2, yellow fever, and West Nile virus capping enzymes. We obtained similar KD values for ppAGUAA bound to the dengue and West Nile virus proteins  . The HilThe flavivirus capping enzyme does not have structural homology to known GTases but does have significant structural homology to methyltransferase enzymes. We performed a TopMatch structural alignment with the Vaccinia virus VP39 methyltransferase protein (PDB code: 1AV6) Based on the structural alignment of VP39 and the dengue virus capping enzyme, we performed a mutagenesis analysis of the dengue capping enzyme to evaluate individual residue contributions to RNA binding. Protein∶RNA interactions commonly occur between basic residues (Arg and Lys) interacting with phosphates within and at the end of the RNA. The region between the GTP and SAM binding site on the capping enzyme is rich with basic residues, and has been hypothesized to be the RNA binding site. To examine the contribution of residues to RNA binding, we individually mutated conserved and semi-conserved (eg. K/R) residues on the GTP/SAM binding face of the capping enzyme and determined how each mutation affected binding affinity di-, mono-, and unphosphorylated RNAs .D . The reD of ppAGUAA in presence of increasing amounts of GTP, GDP, GMP, and ATP (D = ∼18 µM), whereas GDP had a moderate affect on ppAGUAA binding binding (KD = ∼3 µM) and GMP had a very minor effect on ppAGUAA binding (KD = ∼800 nM). The effect of GTP and ATP on ppAGUAA RNA binding (an 80 fold reduction in binding) as compared to GDP and GMP levels suggest that the γ- and β-phosphates on the nucleotides compete with the diphosphate on the RNA for binding. To further examine if the GTP β- and γ- phosphates compete with diphosphorylated RNA binding, we determined the affinity of AGUAA RNA in the presence of 50 µM GTP. We observed that AGUAA binding was weakened 2.4 fold in the presence of 50 µM GTP (KD (Mock) = 3.5 µM, KD (50 µM GTP) = 8.4 µM), indicating that GTP had only minor effects on AGUAA binding. These results suggest that the GTP phosphates interfere with the diphosphate on the ppAGUAA RNA during binding.Diphosphorylated RNA is one of three ligands involved in the guanyltransferase and methyltransferase reactions, the other two being GTP and SAM. We assessed the effects of GTP and SAM on RNA binding to wild-type dengue capping enzyme to determine if either could positively or negatively affected binding affinity. We first determined the K and ATP . We obseD of ppAGUAA binding to the dengue capping enzyme in the presence of increasing amounts of SAM or SAH (D = ∼200 nM), but we did observe a slight increasing in ppAGUAA RNA affinity in the presence of increasing concentrations of both SAM and SAH. A slightly stronger effect on RNA binding affinity was evident with SAM than SAH, suggesting that SAM was able to stabilize RNA binding slightly better than SAH.To examine the effect of SAM and the post-methylation product SAH, we determined the KM or SAH . At low Based on our biochemical data, we mapped residues that significantly interacted with RNAs on RNA binding residues on the dengue virus capping enzyme in complex with GTP (PDB code: 2P1D). In this study we performed a detailed characterization of the RNA binding characteristics of the dengue 2 capping enzyme. We present data demonstrating that dengue, yellow fever, and West Nile virus capping enzyme proteins bind 5′ diphosphorylated end of the viral RNA with similar affinity, although yellow fever virus capping enzyme bound RNA with 2-fold weaker affinity than dengue or West Nile virus capping enzymes. The yellow fever virus capping enzyme has an arginine at position 30 whereas the dengue and West Nile virus capping enzymes have a lysine at position 30, which may explain the small difference in affinity between the viruses . Based oThe overlap of the Vaccinia VP39 protein with the dengue capping enzyme is very strong in the methyltransferase region of the capping enzyme as had been previously noted, and superposition and alignment of residues involved in RNA cap binding have been described Mutation of K62 in the dengue capping enzyme did not affect RNA binding, whereas the homologous lysine residue in the Vaccinia virus VP39 did interact with a phosphate . K181 muAddition of GTP and ATP to the ppAGUAA-FAM binding experiments showed that the phosphates on the nucleotides are able to compete with diphosphorylated RNA binding, and that reducing the number of phosphates on the nucleoside reduces the observed competitive effect. We also observed that high concentrations of GTP had only minor effects on the binding of an unphosphorylated AGUAA RNA, suggesting that GTP predominately interferes with diphosphate binding. The position of GTP binding to the capping enzyme is well known These studies provide a solid foundation for further exploration into how the flavivirus capping enzymes bind to the 5′ diphosphorylated end of the viral genomic RNA during RNA capping. It provides a starting point for further investigations into to determinants of RNA binding, such as further clarifying which sidechains interact with which phosphate, determining the role of ribose hydroxyls in binding, and the specificity for guanine versus adenine in the first nucleotide position during capping


Hepatitis C virus (HCV) genome multiplication requires the concerted action of the viral RNA, host factors and viral proteins. Recent studies have provided information about the requirement of specific viral RNA motifs that play an active role in the viral life cycle. RNA regulatory motifs controlling translation and replication of the viral RNA are mostly found at the 5' and 3' untranslated regions (UTRs). In particular, viral protein synthesis is under the control of the internal ribosome entry site (IRES) element, a complex RNA structure located at the 5'UTR that recruits the ribosomal subunits to the initiator codon. Accordingly, interfering with this RNA structural motif causes the abrogation of the viral cycle. In addition, RNA translation initiation is modulated by cellular factors, including miRNAs and RNA-binding proteins. Interestingly, a RNA structural motif located at the 3'end controls viral replication and establishes long-range RNA-RNA interactions with the 5'UTR, generating functional bridges between both ends on the viral genome. In this article, we review recent advances on virus-host interaction and translation control modulating viral gene expression in infected cells. Flaviviridae family, responsible for non-A non-B hepatitis, affecting about 3% of the human population worldwide. HCV causes persistent infection leading to severe liver damage, including cirrhosis, hepatic steatosis and hepatocellular carcinoma. The virus is predominantly transmitted through the parenteral route during contaminated blood transfusions as well as needle sharing among injecting drug users. Currently, there is no vaccine against HCV. Medical treatment of infected patients with pegylated interferon (IFN)-a and ribavirin is effective only in 40% of the infected population. More recently, a combination of this therapy with viral protease-inhibitors has improved virus clearance, reaching 60% of the HCV patients [Hepatitis C virus (HCV) is a member of the Hepacivirus genus, within the patients . Howeverpatients . It is wpatients ,6. This patients . There apatients .The viral genome consists of a single-stranded RNA of positive polarity, about 9.5 kb in length . UntransIn common with other single-stranded positive-sense RNA viruses, the 5' and 3'UTRs of HCV contain essential structural motifs that exert critical regulatory functions, controlling viral RNA translation and replication ,11. The 7GpppN (cap) at the 5'end of the mRNA is recognized by the eukaryotic initiation factor (eIF)4F complex -binding protein (PABP)). This complex, bound to the 40S small ribosomal subunit, scans the 5'UTR of mRNA in 5' to 3' direction until an initiation codon is encountered in the appropriate context [Most eukaryotic mRNAs initiate translation using a cap-dependent mechanism, by which the m context . In cont context , using a context .Alignment of RNA sequences obtained from clinical isolates corresponding to the HCV IRES region indicated that, though variable, this is the most conserved region of the genome . This fePicornaviridae family that possess HCV-like IRES elements [The HCV IRES spans a region of approximately 340 nt that encompasses most of the 5'UTR of the viral mRNA and the first 30–40 nt of the core-coding region . Variantelements . Given the critical role of the IRES element during HCV multiplication cycle, the RNA structural organization as well as the function of the distinct HCV IRES domains has been analyzed extensively. Domain II consists of two subdomains, IIa and IIb . The RNADomain III is the larger domain of HCV IRES ; it consi are recruited to form the 48S initiation complex [From the mechanistic point of view, binding of the HCV IRES to the 40S ribosomal subunit is the first step of translation initiation on the viral RNA ,42. As m complex . In full complex ,47,48,49 complex ,50. This specific mode of internal initiation of translation is not unique to HCV; HCV-like IRES elements with similar eIF requirements have been found in the genome of some picornaviruses ,52 as weBesides this structural and mechanistic conservation between pestiviruses and the HCV-like IRES of picornaviruses, there are features conserved with more distant IRES elements. Indeed, cryo-electron microscopy studies of the IRES elements of HCV and the intergenic region (IGR) of dicistroviruses  showed that these RNAs are accommodated in the interface of the ribosomal subunits in a similar manner ,53. FurtThe IRES property of interacting with the ribosome is an attractive possibility to explain internal initiation of translation, which is supported by the finding that IRES activity is differentially sensitive to changes in ribosome composition . AnotherBesides ribosomal proteins and eIFs, RNA-binding proteins that play important roles in gene expression help to promote IRES-dependent translation acting as IRES-transacting factors (ITAFs) . Indeed,In addition to eIFs, ribosomal subunits and RNA-binding proteins, HCV subverts the liver-specific microRNA miR-122 to upregulate viral RNA abundance . miR-122in vitro using naked RNA.miR-122 stimulates HCV IRES-mediated translation . In agreThe large difference in the mechanism used by the HCV RNA to initiate translation makes this step of the viral multiplication cycle an ideal target in the development of antivirals. With this aim, many investigators have explored the inactivation of the virus by the development of therapeutic approaches involving direct targeting of the IRES region as well as the interference of specific host factors required for viral RNA translation. Attempts to block IRES activity directly with antisense oligonucleotides, peptide nucleic acids (PNAs), locked nucleic acid (LNAs), DNA and RNA aptamers, RNAi and ribozymes have been widely explored ,78,79,80Screening of small molecule inhibitor libraries has yielded limited success to control HCV multiplication . Howeverin vitro, using cell-free systems or tissue culture cells, the lack of a convenient laboratory animal model has hampered the development of effective therapies. Interestingly, expression of two human genes, CD81 and occludin, has been shown to be sufficient to allow HCV infection of fully immunocompetent inbred mice [Although many attempts to inhibit different steps of the HCV multiplication cycle have been done red mice . This ancis-acting replication element (CRE)  , a strucA functional role of the IRES-5BSL3.2 long-range RNA interaction in translation control has been recently documented . MutatioGenome circularization promoted by direct RNA-RNA contacts involving inverted complementary sequences has been shown to be essential for viral replication in the case of flaviviruses . In otheIn the search of factors that could generate functional bridges between the ends of the HCV viral genome, it was found that the insulin-like growth factor II mRNA-binding protein 1 (IGF2BP1) is present in complexes assembled with RNAs that contained both the HCV IRES and the 3'UTR. This protein coimmunoprecipitates with eIF3 and the 40S ribosomal subunit  suggestii ternary complex, and thus inhibiting mRNA translation [i to the P site of the 40S ribosomal subunit [2 in cell free systems [HCV, as all RNA viruses, depends on the host cell machinery to synthesize the proteins necessary for viral replication. Consequently, RNA viruses have developed specialized strategies to subvert the cellular machinery in their own benefit, including cellular translation factors and signaling pathways that control host protein synthesis . In turnnslation . To coun subunit . Further systems ; whetherAn alternative way used by RNA viruses to control the translation repression effect induced by the host stress response to infection is to take advantage of the oscillation in this response to prevent continuing translation repression . Virus iHCV-induced persistent infection reflects the evasion of host immunity and interference with IFN innate immune defenses. Cellular antiviral immunity, among others, is based on the host recognition of virus dsRNA intermediates, which are sensed by Toll-like receptor 3 (TLR3) and the RNA helicase RIG-I (retinoic acid inducible gene I). Detection of dsRNA results in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB. CARD-adaptor proteins interact with RIG-I and recruits IKK kinases, leading to the activation of NF-kappaB and IRF3. However, the HCV NS3-4A viral serine protease cleaves cardif   as well The response of the cell to HCV infection affects various pathways that may differ between individual patients, determining the outcome of the infection. In fact, both cytopathic effects and immune-mediated processes play an important role in liver injury. At least in part this has been illustrated by cell lines that have different permissiveness to infection and show a differential expression of genes linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication . On the Understanding the biological role of RNA regulatory elements present in the HCV genome has received great attention in the last years. In particular, the concerted action of these signals alongside host and viral factors is essential to control the viral multiplication cycle and at the same time to maintain the permissiveness of the cell to a sustained persistent infection. Mechanistic aspects that explain at the molecular level how HCV RNA structures and viral proteins participate in the infection process still need to be investigated in detail. In particular, efforts towards identifying functionally relevant interactors of the viral RNA regulatory elements, as well as viral proteins, will provide information about their mechanism of action. An important question is how these functional structures control the switch from translation to replication and later to encapsidation of the viral genome. In recent years, it has become evident that dynamic conformational changes controlled by RNA-RNA, RNA-protein and miRNA-RNA interactions modulate viral replication in the viral genome. This view of the viral genome as a flexible molecule will help in understanding the function of RNA structures formed at different stages of the viral life cycle. Deciphering how these high order structures are assembled in the context of the whole viral genome and how these macromolecular complexes govern the viral multiplication cycle is ahead of us.


Escherichia coli via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (24LYDLDIESPGHEQK37) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5′-32P-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (427LIEEEALK433), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (65HGFLPL*K71). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E in vivo. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in  Escherichia coli, RNase E  is an essential protein with a mass of 118.2 kDa and a subunit structure that consists of two distinct halves. The N-terminal domain  contains the site-specific endonuclease activity that is sufficient for cell survival RNase E participates in the degradation and processing of RNAs in bacterial cells. Although RNase E has a preference for the cleavage of single-strand AU-rich regions within RNA substrates, the enzyme efficiency and specificity for the cleavage site seems to be affected by the stem-loop structure, membrane binding and phosphorylation status at the 5′ terminus of RNA substrates E. coli. Adventitious overexpression or increased enzymatic activity of RNase E cause cellular toxicity leading to growth retardation The enzymatic activity and cellular concentration of RNase E are strictly regulated through various mechanisms in 24LYDLDIESPGHEQK37 peptide region in vivo and in vitro, in contrast to the effects of the Q36R mutant. Using a radiolabeled BR13 oligoribonucleotide (p-BR13) substrate, EMSA, UV crosslinking and mass spectrometry analyses were performed to examine the molecular interactions between RNA and RNase E. This study shows that the RNase E has two alternative RNA binding sites at the N-terminal domain, which contribute to the regulation of the enzyme activity by uncompetitive and allosteric inhibition modes.From mutagenesis studies, a hyperactive mutant with a substitution of glutamine to arginine at position 36 (Q36R) has been found to enhance RNase E activity due to a decrease in uncompetitive inhibition of the RNA substrate bound to the NarI and HindIII , and cloned into compatible sites of pNRNE4. The primers used were Nrne1-F (5′-ACCACCCTGGCGCCCAATACGCAA-3′), Nrne1-R (5′-ATATCCAGGTCAGCCAGACGCTGCC-3′), Nrne2-F (5′-GGCAGCGTCTGGCTGACCTGGATAT-3′), and Nrne2-R (5′-TTTCAGACGGAAGCTTAAATCCCA-3′). The pLAC-RNE2-Y25A plasmid was constructed by subcloning the NarI and HindIII fragment of pNRNE-Y25A into the same restriction enzyme sites in pLAC-RNE2.The strains and plasmids used in this study are listed in 32P]-ATP using T4 polynucleotide kinase  and the labeled products (p-BR13) were purified by MicroSpin™ G25 columns  according to the manufacturer's instructions 2, and 50% (v/v) glycerol. The reaction products were separated in 15% denaturing polyacrylamide gels.BR13 was 5′-end labeled with .in vitro, 5′-32P-end-labeled BR13 (p-BR13) was utilized as the substrate, and the cleavage products were resolved on PAGE gels and analyzed by autoradiography (In order to assay the enzymatic activity of wild-type and mutant N-Rne proteins iography . When thKD) was determined by measurements of the free to bound RNA concentration ratio, as described previously KD value of the Y25A mutant (17.6 µM) was determined to be 1.2-fold higher than that of the wild-type N-Rne, (14.6 µM), and the Q36R mutant exhibited a low KD (6.5 µM) compared to that of the wild-type N-Rne and the Y25A mutant  were performed by mixing various concentrations of the purified proteins with a fixed concentration of p-BR13. To estimate the protein-RNA complex strength, the dissociation constant   to determine whether the mutations that alter the binding and enzymatic activity of the mutant N-Rne proteins lead to the misfolding of the protein. As shown in Figure S3 in To compare RNA binding potentials, extracted ion chromatograms (XICs) of the parental and nucleoside-bound peptides were generated by normalizing the signal intensity with the peak of a unique stripped peptide, 24LYDLDIESPGHEQK37 peptide includes an alternative RNA binding site of RNase E at the Y25 and Q36 residues bonded with cytosine and adenine, respectively  and the corrected PSM levels . A unique stripped peptide,16VALVDGQR23, used as the internal standard (IS) for calculation of relative intensity or corrected PSM level was included as below. (A) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of the stripped IS peptide, 16VALVDGQR23, at m/z = 429.5 in a charge state of +2. (B) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of a parental RWT peptide of the wild-type N-Rne, 26DLDIESPGHEQK37, detected at m/z = 684.7 in the charge state of +2. (C) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of the RWT* peptide, 26DALCDIESPGHEQK37, detected at m/z = 627.0 in the charge state of +3 with the D26 and L27 residues bound to adenine (A) and cytosine (C), respectively. (D) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of a parental RY25A peptide of the Y25A mutant, 24LADLDIESPGHEQK37, detected at m/z = 776.6 in the charge state of +2. (E) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of a parental RQ36R peptide of the Q36R mutant, 26DLDIESPGHER36, detected at m/z = 634.5 in the charge state of +2. (F) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of a parental P peptide, 65HGFLPLK71, detected at m/z = 406.3 in the charge state of +2. (G) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of the P* peptide, 65HGFLPUKAL71, detected at m/z = 662.2 in the charge state of +2 with the L70 and K71 residues bound to uracil (U) and adenine (A), respectively. (H) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of a parental M peptide, 427LIEEEALK433, detected at m/z = 473.0 in the charge state of +2. (I) Tandem mass spectrum assigned to the predicted b- and y-ions generated from collision-induced fragmentation of the M* peptide, 427LIEEEALKC433, detected at m/z = 594.5 in the charge state of +2 with the K433 residue bound to cytosine (C). Figure S3. Detection of misfolding of N-Rne mutants. Purified proteins of N-Rne, N-Rne-Y25A, and N-Rne-Q36R were used to measure the CD spectrum. Table S1.Results of nLC-tandem mass spectrometry analyses of wild-type N-Rne, Y25A and Q36R mutant samples crosslinked with p-BR13.Note. Variable modifications at the positions with the lowercase one-letter amino acid codes: CAM, carbamidomethylation (C); O, oxidation ; and UV crosslinked residues with nucleosides  in parentheses.(DOCX)Click here for additional data file.