Caenorhabditis elegans homolog of the giant muscle protein obscurin, UNC-89, is required for normal muscle cell architecture. The large immunoglobulin domain-rich isoforms of UNC-89 are critical for sarcomere and sarcoplasmic reticulum organization. Furthermore, we have found evidence that this structural organization is crucial for excitation-contraction coupling in the body wall muscle, through the coordination of calcium signaling. Thus, our data implicates UNC-89 in maintaining muscle cell architecture and that this precise organization is essential for optimal calcium mobilization and efficient and effective muscle contraction.Calcium, a ubiquitous intracellular signaling molecule, controls a diverse array of cellular processes. Consequently, cells have developed strategies to modulate the shape of calcium signals in space and time. The force generating machinery in muscle is regulated by the influx and efflux of calcium ions into the muscle cytoplasm. In order for efficient and effective muscle contraction to occur, calcium needs to be rapidly, accurately and reliably regulated. The mechanisms underlying this highly regulated process are not fully understood. Here, we show that the  Muscle contraction is triggered by the rapid entry of calcium, a ubiquitous and diverse signaling molecule, into the muscle cytosol. Thus, cytosolic levels of calcium need to be reliably and accurately regulated both spatially and temporally for effective muscle contraction. The sacromere contains several giant proteins, including titin, nebulin and obscurin, which are hypothesized to be critical components involved in sarcomere assembly and organization. While both titin and nebulin have been postulated to be molecular scaffolds that have roles in dictating the length of myofilaments In addition to its localization to the M-line, obscurin associates with the sarcoplasmic reticulum (SR), a key storage organelle for calcium. This association is mediated by a direct interaction of obscurin with the SR membrane protein small Ankyrin 1 C. elegans genome contains only one obscurin homolog, UNC-89, which like obscurin localizes to the M-line and has been implicated in myofilament assembly and organization C. elegans muscle.Unlike mammals, the C. elegans. Over-expression of vav-1 (accession number Q45FX5), a Rac GTPase family guanine nucleotide exchange factor, in the body wall muscle leads to erratic calcium signaling and uncoordinated locomotion vav-1 is over-expressed in body wall muscle cells, the animals display a disrupted sinusoidal pattern and slow rate of locomotion (egl-19(tak5) and a loss of function allele of unc-89(ak155) , which encodes an L-type voltage gated calcium channel (L-VGCC) unc-89 (accession number O01761) was not anticipated. Additionally, we have found that a previously isolated reduction of function allele of egl-19, n582unc-89, e1460vav-1 over-expression (data not shown).The isolation of a mutation in egl-19(ad695gf) that has been shown to produce a persistent influx of calcium into body wall muscle cells egl-19(gf) mutant animals display a short hyper-contracted body phenotype as compared to wild-type animals (unc-89(e1460) or unc-89(ak155) mutations into egl-19(gf) mutants would suppress the short hyper-contracted body phenotype associated with the increased calcium signaling in these mutants. Consistent with the suppression of vav-1 over-expression, we found that introduction of unc-89(ak155) or unc-89(e1460) mutations into egl-19(gf) mutants can suppress the hyper-contracted short body phenotype of egl-19(gf) mutants  and unc-89(e1460) mutants, we sequenced the unc-89 genomic region in these animals. We identified nonsense mutations in both unc-89(ak155) and unc-89(e1460) mutants that introduce stop codons in UNC-89 at Ig repeat 15 and 21, respectively (egl-19(gf), we utilized unc-89(st79), which contains a stop codon in the Ig domain that lies just N-terminal of the second protein kinase domain (unc-89(ok1116), which contains a deletion of the second kinase domain (unc-89(st79) no longer expresses the large UNC-89B and UNC-89F isoforms egl-19(gf) background, neither can suppress the hyper-contracted short phenotype of egl-19(gf)  , suggestpression , while Reraction .C. elegans genome does not encode a TC10 homolog and the closest relative to TC10 in C. elegans, CDC-42, does not interact with the Dbl homology domain of UNC-89 rho-1 in an attempt to suppress egl-19(gf) or over-expression of vav-1. rho-1(RNAi) treatment failed to suppress the egl-19(gf) short hyper-contracted body phenotype  is expressed and Obsl1, an Ig domain-rich protein, is normally localized to the M-line in the obscurin knock out mice C. elegans genome does not encode an apparent Obsl1 homolog, we examined myofilament assembly and organization in unc-89 mutants.Previous studies on unc-89 mutants. Surprisingly, myosin filaments assembled normally in unc-89(ak155) and unc-89(e1460) larvae and were indistinguishable from wild-type larvae (unc-89(ak155) and unc-89(e1460) mutants, their organization is lost as the mutant animals reach adulthood. In wild-type animals, anti-myosin immunofluorescence labels approximately 10 A-bands per muscle cell that run longitudinally in the antero-posterior axis  mutants indicates clearly formed thick and thin filaments; however, while the filaments are closely aligned, their organization is scattered in the myoplasm As shown in ial lawn . This suunc-89 mutants exhibit fairly normal sinusoidal locomotion, they displayed a reduced level of body bends compared to wild-type animals, indicating that muscle function is compromised in unc-89 mutants , which is implicated in the regulation of actomyosin interactions during contraction unc-22(RNAi) is used to knock down UNC-22/twitchin in egl-19 mutants  mutants (unc-89(ak155) mutants, the distribution of SERCA is disorganized  mutants revealed reliable calcium transients in both the pharyngeal muscle ; however, the time to reach baseline (decay time) was not significantly different from wild-type animals  I, unc-89(e1460) I, unc-89(st79) I, unc-89(ok1116) I, egl-19(tak5) IV, egl-19(n582) IV, egl-19(ad695) IV, unc-22(e66) IV, rho-1(ok2418) IV, unc-68(r1162) V, akIs42 [Pmyo-3::vav-1], akIs104 (Pmyo-3::YC3.60), akIs106 (Pmyo-2::YC3.60), sca-1::GFPcimEx4(egl-19::mCherry)zwIs108 (Pmyo-3::MYC::unc-68)Nematodes were grown at 20C and the vav-1 in the body wall muscle (akIs41) was mutagenized with 50 mM ethylmethane sulfonate using standard procedures egl-19(tak5) and unc-89(ak155). These mutant loci were mapped to their chromosomal position by single nucleotide polymorphism mapping using standard procedures ak155)(details below) or rescue of mutant phenotype (tak5) with cimEx4(egl-19::mCherry)In two separate mutagenesis screens, a strain overexpressing C. elegans genomic sequence obtained from WormBase.org (Oct. 2011), using stringent parameters (0.7 on the length and 0.8 similarity), yielding an average coverage of >50X. SNPs and InDels were identified with minimum coverage requirements of >25X. Our analysis was then focused on the ∼60 kb unc-89 gene. For each mutant strain, a single homozygous mutation in the unc-89 coding sequence was identified. Each of these candidate mutations was verified by using the Sanger method to sequence ∼600 bp segments PCR-amplified from single worms of each strain.Genomic DNA from each strain was prepared and shotgun libraries were prepared following instructions from Illumina. The entire genomic sequence from each strain was determined by sequencing shotgun clones on an Illumina HiSeq with 100 base reads and the program SCS2.8 was used to process the run and call the bases. The sequence files were imported into CLC Genomics Workbench 4.8 and trimmed to </ = 1 ambiguous bases with a minimum read length of 36 bases. The trimmed reads were mapped against the myo-3 promoter vector, pPD95.86 (Addgene). Similarly, Pmyo-2::YC3.60 construct was assembled by PCR amplifying YC3.60 with primers containing NheI and Acc65I restriction sites. The PCR amplified YC3.60 was digested with NheI and Acc65I and cloned into the vector containing the myo-2 promoter vector, pPD94.48 (Addgene). Using standard procedures lin-15(+)] were injected into lin-15(n765ts) animals. Transgenic progeny were identified by body wall or pharyngeal muscle fluorescence and lin-15 mutant rescue. Stable integrated lines were generated with X-rays using standard procedures Pmyo-3::YC3.60 construct was assembled by PCR amplifying YC3.60 (kind gift from Atsushi Miyawaki) with primers containing NheI and Acc65I restriction sites. The PCR amplified YC3.60 was digested with NheI and Acc65I and cloned into the vector containing the Images were taken on a Zeiss Discovery V12 dissecting microscope with a 3.5× PlanApo S objective using a Canon Powershot G11 digital camera of young adult animals . Images were processed and analyzed using ImageJ. Body size was determined by length  divided by width . Additionally, tip of the nose to the posterior edge of the pharyngeal terminal bulb (mm) was measured to investigate hyper-contraction of the body.E. coli [HT115 (DE3)] containing an RNAi construct specific to the kinase containing isoforms of UNC-89 (JA:C24G7.5) rho-1rho-1(RNAi) experiments, since exposure of L4 animals for 24–36 hours to rho-1(RNAi) results in ∼100% embryonic lethality in the F1 progeny, L1 larvae were grown and allowed to feed on bacteria carrying an RNAi construct specific rho-1 for approximately 48 hours rho-1(RNAi) treated animals was carried out once these animals developed into young adults. RNAi knockdown was verified by observing reported phenotypes in RNAi treated animals L4 animals were grown and allowed to feed on Animals were fixed in Bouin's fixative for one hour at room temperature. After the initial 30 minutes the animals were freeze cracked by incubation in an ethanol dry ice bath for two minutes and rapidly thawed in hot tap water. Fixed animals were thoroughly washed in borate buffer . Antibodies were added in standard phosphate buffer saline containing 0.5% Triton. Antibodies used were a myosin heavy chain A antibody, DM5.6,  Pmyo-2::YC3.60 were immobilized in M9 containing 1 mM levamisole and 10 mM 5-hydroxytryptamine. For calcium imaging in the body wall muscle, animals expressing Pmyo-3::YC3.60 were immobilized using cyanoacrylate surgical glue (3M) and dissected on a polydimethylsiloxane (Sylgard) coated cover slip in extracellular fluid . The intestine and gonad are removed by mouth suction pipette and the basement membrane is removed by collagenase and proteinase K treatment for 30 seconds. To apply ACh (100 µM), a pipette was placed close to the body wall muscle and applied by pressure ejection to deliver a short duration of ACh (∼100 millisecond). Images were collected on a Zeiss AxioObserver equipped with an optical beam splitter (Photometrics) and a Hamamatsu ORCA-R2 charged-coupled device camera using Zeiss AxioVision software. Image analysis was carried out in AxioVision using the physiology module and exported to Excel (Microsoft) for presentation and Prism (GraphPad) for statistical analysis.For calcium imaging in the pharynx, animals expressing L4 animals of each genotype were picked to fresh OP50 plates and allowed to mature at 20°C for 24 hours. The swimming assays were carried out on individual young adult animals in M9 by counting complete body deflections for 30 seconds. For locomotion (crawling), body bends of individual young adult animals were counted for 1 minute. A body bend was scored as a complete deflection of the anterior portion of the nematode from the midline. 20 animals of each genotype were analyzed. Animal velocity was determined by video microscopy tracking of multiple worms using an Olympus SZ61 stereomicroscope equipped with an Sony SXD-900 charge-coupled device camera. Movies were taken and Velocity was calculated using MatLab (Mathworks) software Statistical Analyses were carried out in Excel (Microsoft) or GraphPad (Prism). Data was presented as means ± SEM. Statistical significance was determined using a Student's two tailed t test or ANOVA followed by Dunn's multiple comparison test. P values<0.05 were taken to indicate statistical significance.Figure S1The large Ig domain-rich isoforms of UNC-89 are required for the egl-19(gf) induced hyper-contracted body phenotype. Tip of the nose to the posterior edge of the pharynx measurements were determined. egl-19(ad695gf) animals were hyper-contracted compared to wild-type animals (* p<0.05). unc-89(ak155) or unc-89(e1460) into the egl-19(ad695gf) background significantly reduced the hyper-contracted body phenotype (**p<0.05), whereas unc-89(st79) and unc-89(ok1116) had no affect on the egl-19(ad695gf) hyper-contracted body phenotype. n = 10 for each genotype.(TIF)Click here for additional data file.Figure S2The large Ig rich isoforms of UNC-89 are required for normal localization of SERCA. (A) Representative image of SERCA::GFP localization in a wild-type animal. (B) Representative image of SERCA::GFP localization in a wild-type animals treated with RNAi specific to the kinase containing isoforms of UNC-89. (C) Representative image of SERCA::GFP localization in a wild-type animals treated with RNAi specific to the large Ig domain-rich isoforms. Note the disorganization of the linear punctate structures in the wild-type animals treated with the RNAi specific to the large Ig domain-rich isoforms of UNC-89. Scale bar = 5 µm.(TIF)Click here for additional data file.Table S1Shows the sequence alteration in the unc-89 coding region in unc-89(ak155) and unc-89(e1460) mutants.(DOCX)Click here for additional data file.Video S1Shows wild-type locomotion.(MOV)Click here for additional data file.Video S2Shows uncoordinated locomotion of animals over-expressing vav-1.(MOV)Click here for additional data file.Video S3Shows wild-type like locomotion of egl-19(tak5) mutant animals over- expressing vav-1.(MOV)Click here for additional data file.Video S4Shows wild-type like locomotion of unc-89(ak155) mutant animals over- expressing vav-1.(MOV)Click here for additional data file.


The discovery of discontiguous tRNA genes triggered studies dissecting the process of tRNA splicing. As a result, we have gained detailed mechanistic knowledge on enzymatic removal of tRNA introns catalyzed by endonuclease and ligase proteins. In addition to the elucidation of tRNA processing, these studies facilitated the discovery of additional functions of RNA ligases such as RNA repair and non-conventional mRNA splicing events. Recently, the identification of a new type of RNA ligases in bacteria, archaea, and humans closed a long-standing gap in the field of tRNA processing. This review summarizes past and recent findings in the field of tRNA splicing with a focus on RNA ligation as it preferentially occurs in archaea and humans. In addition to providing an integrated view of the types and phyletic distribution of RNA ligase proteins known to date, this survey also aims at highlighting known and potential accessory biological functions of RNA ligases.The online version of this article (doi:10.1007/s00018-012-0944-2) contains supplementary material, which is available to authorized users. Transfer RNAs (tRNA) are transcribed as precursor transcripts and are subjected to a series of posttranscriptional processing events before they are matured to fulfill their biological functions , 2. SequIn contrast to bacterial pre-tRNAs—which are disrupted by self-splicing group I introns , 13—archIn archaea, as many as 70% of genetically encoded pre-tRNAs can be disrupted by introns of 16–44 nucleotides in length, which are characterized by the presence of a highly conserved “bulge-helix-bulge” motif , 15. In Cyanidioschyzon merolae , α2 , (αβ)2 , and αβγδ  enzymes [Studies modifying the substrates used for in vitro processing reactions revealed that splice site recognition by archaeal tRNA splicing endonucleases relies on the presence of the bulge-helix-bulge motif . Archaea enzymes , 42–44.  enzymes . The monScSEN2 and ScSEN34, and two structural subunits, ScSEN15 and ScSEN54 [HsTSEN2, HsTSEN34, HsTSEN15, and HsTSEN54 [The eukaryal tRNA endonucleases are phylogenetically related to their archaeal counterparts . The yea ScSEN54 . All fouHsTSEN54 . AlthougHsTSEN54 , eukaryaHsTSEN54 . Apart fHsTSEN54 , little HsTSEN54 .Escherichia coli has been shown to catalyze ligation of tRNA exon halves [E. coli [Proteins catalyzing the final step of tRNA splicing—ligation of tRNA exon halves —have firn halves . Even ea[E. coli . These e[E. coli   acts both as phosphatase [cis diol and 5′-phosphate termini  subsequently joins these tRNA halves by 5′–3′ RNA ligation thus restores a functional tRNA [Several bacterial species have evolved to sacrifice individual members of their populations upon phage infection by activating various suicide response pathways . One of  tRNALys . It has sphatase  and polysphatase  thus connal tRNA .In addition to T4 Rnl1, another RNA ligase, T4 Rnl2, has been identified in the genome of bacteriophage T4 . T4 Rnl1ScTRL1) and plant  tRNA ligases—both identified by activity-guided chromatographic purification [cis diol termini generated by T4 Pnkp  featuring a stand-alone ligase protein with weak homology to yeast and plant RNA ligases [BfKinase/CPD or BfCLP1) do not reside on the ligase polypeptide  [HsCNP) [HsTRPT1) [HsCLP1, is an integral component of the tRNA endonuclease complex, suggesting the occurrence of coupled endonuclease and kinase reactions in humans [S. cerevisiae, where ligase and kinase activities both reside on ScTRL1. However, the relevance of the 5′–3′ RNA ligase mechanism for tRNA splicing in vertebrates is under debate since the 2′-phosphotransferase is not essential in mice [Although a 5′–3′ RNA ligase activity has been detected in biochemical experiments carried out with HeLa cell extracts , no homo ligases . In this HsCLP1) , cyclic  [HsCNP) –91, and HsTRPT1)  activitiHsTRPT1) –95 has tHsTRPT1) . In suppn humans , 88. In  in mice .To account for the substantial differences in sequence and domain organization, it has been suggested to group 5′–3′ RNA ligases in bacteriophages, yeast, plants, and animals into classes , 87; howPyrobaculum aerophilum and in humans [Escherichia coli as a 3′–5′ RNA ligase [Biochemical studies revealed that ligation of tRNA exon halves in vertebrates and archaea is mainly achieved by an alternative mechanism. A 3′–5′ RNA ligase activity resulting in incorporation of the precursor-derived 2′,3′-cyclic phosphate into the splice junction as a 3′,5′-phosphodiester was for the first time detected in HeLa cell extracts .3′–5′ RNA ligation appears to be the prevalent human tRNA splicing pathway , 56 and DDX1 observed in cancerous cell lines [The human tRNA ligase complex components seem to be constitutively and widely expressed in all human and mouse tissues analyzed so far, indicating that these proteins cooperate in a great variety of cellular contexts , 112. DDll lines , 120, itll lines –123, andll lines  are linkPublished information concerning the function of CGI-99 is scarce. Although the protein has been found to interact with human , 125 andThe identification of FAM98B as a human tRNA ligase complex component is the first piece of information published concerning its molecular function. Within the context of tRNA splicing, the exact role of FAM98B needs to be established. Since its RNAi-mediated depletion has almost no impact on RNA ligase activity in HeLa cell extracts , it is lXenopus laevis [X. laevis leads to severe developmental defects, it provided limited insights into mechanistic aspects of ASW function. ASW has been speculated to be specific for vertebrates as no homologous genes were detectable in Drosophila melanogaster or Caenorhabditis elegans [X. laevis.ASW has first been characterized in a genetic screen searching for genes differentially expressed in early neural specification in s laevis . Althoug elegans . NonetheTaken together, apart from HSPC117, which appears to be conserved among all domains of life, the additional components of the HSPC117 complex do not show a comparably broad taxonomic coverage. Orthologues of DDX1, FAM98B, and CGI-99 are spread more widely than ASW, which seems to be restricted to fewer species Fig. A.ligT, conserved not only in bacteria but also in euryarchaeota and crenarchaeota [E. coli catalyses an ATP-independent equilibrium reaction between 2′,3′-cyclic phosphate and 5′-hydroxyl termini and 2′,5′-phosphodiester bonds [Pyrococcus furiosus confirming the broad phyletic occurrence of LigT proteins [Another RNA ligase activity leading to the unusual 2′,5′-phosphodiester bond has been shown to act in extracts prepared from various bacterial species  where an RtcB/HSPC117 protein has been retained [B. floridae 5′–3′ RNA ligase BfRNL [RNA ligase proteins are detectable within all major lines of descent. Considering the different ligation mechanisms and classes of RNA ligase polypeptides, it seems, however, that several species abandoned one or the other mechanism of RNA ligation Figs. B, 6. Mosretained , 128 Below is the link to the electronic supplementary material.


Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking.RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of  RNA trafficking in plants has received increasing attention in recent years. It is an important phenomenon in that it suggests that plants operate at the level of supracellular organisms, meaning that mRNA is not restricted to the cell in which it was synthesized, but rather moves from cell-to-cell and even over long distances through the phloem . Under tHowever, the study of RNA movement between cells of a single organism presents tremendous challenges. It is difficult to confidently determine the cell of origin of an RNA molecule when the genotypes of source and destination cells are identical. This inability to distinguish mRNA origins hampers research, and while it is currently thought that there are about 1,100 proteins and hundreds of mRNAs that undergo long-distance movement , the actCuscuta pentagona), the connections permit transfer of mRNA from host to parasite , and have the ability to transmit RNA  hosts. The host range of parasitic plants may also vary substantially, and some species are able to parasitize a wide range of host species while others are limited to just a single genera. Certain parasite species have adapted to attack relatively broad ranges of crop plants and are economically important agricultural weeds  and broomrapes (Orobanche and Phelipanche spp.), two genera with relatively well-characterized haustoria. RNA trafficking to parasitic plants has been best characterized in these species, particularly Cuscuta, but even here the level of understanding of RNA movement is far from complete.Parasitic plants live by tapping into the vascular system of a host plant and withdrawing the necessary water and nutrients needed to provide part or all of the parasite nutritional needs. Parasites connect to their hosts using a specialized structure, the haustorium, which penetrates host tissue and forms a bridge to the vascular system of the host. Haustoria vary substantially in their anatomy and function among different parasitic plant species, and are generally characterized by whether they form connections exclusively to the xylem only or to both xylem and phloem . Xylem-fCuscuta, the overwhelming physiological data indicates that the parasite absorbs phloem contents from the host. However, the exact mechanism is not clear as no direct phloem connections have been demonstrated  connections with host cells . The capacity of Cuscuta to acquire mRNAs from hosts was first demonstrated for mRNAs of specific genes known to be mobile in pumpkin phloem (Cuscuta stems near the point of host attachment and used in reverse transcriptase (RT)-PCR and the resulting amplified products were then sequenced to confirm that the mRNA sequence detected in the parasite was identical to that of the host gene. Additional experiments used tomato hosts and hybridized RNA extracted from Cuscuta onto a tomato microarray, leading to the identification of 474 putatively mobile mRNAs. Confirmation tests on a subset of these using RT-PCR, along with an additional study  provides evidence that the phenomenon is not specific to just one host–parasite interaction.Parasitic plants acquire a wide range of macromolecules from their hosts that are relevant to the current discussion  revealed no host mRNAs in the shoots of attached P. aegyptiaca, suggesting that host mRNA mobility into this parasite may be limited at the point of haustorial transfer into the parasite or between the tubercle and shoot of the parasite. Further experiments are needed to specifically answer this question.The mobility of host mRNAs into  Cuscuta . More reTable 1). Cuscuta has long been recognized for the ability to accept and transmit viruses with its hosts and in fact has often been used as a vector for transmitting plant viruses between different plants (Cuscuta (N) transmitting through a Cuscuta bridge between two Nicotiana tabacum plants, yet showed little virus accumulation in the Cuscuta bridge itself compared to recipient host plant and suggesting that virus movement occurs in Cuscuta without multiplication in the parasite (P. aegyptiaca the uptake of three positive ssRNA viruses cucumber mosaic virus (CMV), tomato mosaic virus (ToMV), potato virus Y (PVY), and the ssDNA virus tomato yellow leaf curl virus (TYLCV) were shown to move into the parasite from infected hosts and, in the case of CMV, is able to replicate in the parasite  was transformed to express the β-glucuronidase (GUS) reporter gene and then allowed to parasitize lettuce plants that expressed a fragment of the GUS gene in a hairpin orientation  in P. aegyptiaca using constructs expressed in a tomato host  hosts. Cuscuta bridges connecting Alder Yellows (ALY)-infected alder to non-infected periwinkle were able to transfer the phytoplasma, and symptoms were slowly manifested 4 months after the Cuscuta bridge was established (Lilium (hybrid Casablanca) to periwinkle through the Cuscuta bridge, with the recipient plant developing symptoms of stunting and flower bud deficiency 2–3 weeks following connection by Cuscuta . Although HGT and RNA trafficking both involve movement of nucleic acids, it is not certain whether they use similar mechanisms. HGT has been reported between several non-parasitic plants, but seems to occur at a higher frequency in parasitic associations . The higStriga spp. , and these have been proposed to transfer between plants as large sections of mitochondrial DNA  phloem protein 16 (CmPP16) in order to enter the phloem stream , interact with specific RNA binding proteins much like the MPs of RNA viruses (discussed below). The MPs bind RNAs, repress translation, and enable movement of the ribonucleoprotein (RNP) complex to a PD . Mutatiom stream , and reqm stream . Shortrom stream .E. coli KNOTTED1 mRNA transcript was microinjected into maize mesophyll adjacent to vascular tissue, and was seen to migrate from cell-to-cell, presumably via PD connections. Mutated KN1 proteins were microinjected to show that the homeodomain protein was necessary for translocation.One of the best characterized mobile mRNAs is KNOTTED1 (KN1). Transcription factors of the KNOTTED1-like family are ubiquitous plant cell constituents responsible for regulating pattern formation of the apical meristem. The KN1 homeodomain protein complexed to its own mRNA was detected in phloem and was associated with determining cell fate . FluoresFigure 1). We focus on host–parasite connections via a shared PD because in the case of Cuscuta there is both physical and experimental evidence for trafficking through PD (discussed above) and for broomrapes the PD may initiate the formation of sieve pores between species  to facilitate passage through PD . Two modStBEL5 fused to the coding sequence of a less mobile BEL-homolog increased mobility  RNA, which travels by means of a cis-acting element on the transcript, independent of the essential FT protein, from leaf to shoot apical meristem to induce flowering and suggests that mRNA has a role in systemic signaling of major developmental transitions , 2011. IStudying RNA mobility over short and long distances in plants is technically challenging because of the need to pinpoint source and destination cells of the mRNA. Techniques range from the elegant application of transgenics, phloem-sap collection (either directly or using aphid stylets), grafting, or combinations of these . Each apStudies of short-distance movement of mRNA have relied on transgenics and the use of genes with high specificity of expression , microinInvestigations of long-distance trafficking are somewhat simpler because RNAs identified from phloem sap are assumed to be in the process of moving from source to destination. Direct collection of phloem sap exuded from stem incisions is the ideal, but relatively few herbaceous species are copious exuders like cucurbits  and leguAnother ingenious approach for sampling phloem is aphid stylectomy, which uses the ability of aphids to precisely insert their stylets into plant sieve elements. Subsequent severing of the aphid from its stylet leaves a tube that exudes small droplets of nearly pure phloem sap . DisadvaStBEL5  between related species such as pumpkin and cucumber have been used in several key studies of RNA trafficking . SolanacStBEL5 , and than potato  and the n potato . A procetely 11% . A disadtely 11% , which lArabidopsis , the process of withdrawing host vascular contents is likely within the range of normal plant translocation.A concern over analyses of phloem sap obtained from incisions is that the sudden release in pressure would create artifacts such as dislodged macromolecules that would otherwise not be mobile in sieve elements . ParasitCuscuta connections have certain disadvantages. Among these are the technical issues of establishing connections on specific host locations and at specific times. Parasites are somewhat unpredictable, and generating uniform tissues from synchronized attachment points can be challenging. In Cuscuta, the haustoria also follow a developmental progression through stages of host penetration, vascular connection, and eventually occlusion . On the other hand, if trafficked RNA acts in a sequence-specific manner, then function in a parasite would depend on the presence of sufficiently homologous genes in the parasite. Many trafficked mRNA may not have a metabolic or regulatory function and may simply serve as a nutrient source for the parasite. Of course all three of these possibilities may occur, depending on the transcript in question.The most intriguing questions regarding cross species movement of RNA are whether and how RNA from one species functions in another species. Answering this question is difficult because mechanisms of action of native trafficked RNAs are not fully understood. If mRNAs function through translation into protein, as has been indicated for KN1 , then paCuscuta flowering time depends on timing of host flowering, thus invoking the possibility that FT RNA and protein from the host are capable of inducing flowering in the parasite , DNA mismatch repair protein (Solyc02g082660.2.1), clathrin heavy chain (Solyc06g051310.2.1), and ATPase subunit 1 (Solyc11g039980.1.1) are 80% (464/583), 83% (166/199), 85% (720/852), and 96% (251/261) identical, respectively, at the nucleotide level between tomato and C. pentagona  show evidence of HGT events in phylogenetic studies (Table 1). However, the functional significance of most HGT events involving parasites is unclear and a benefit to the parasite has not yet been demonstrated for any gene acquired in this way.There may be an evolutionary benefit to trafficking of nucleic acids into the parasite, such as for horizontal gene transfer . Many paC. pentagonaPYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE β subunit mRNA was negative (Finally, it is interesting to speculate whether RNAs from the parasite could be used as pathogenic factors in establishing and maintaining host connections. Although an assay for parasite-to-host movement of a negative , the movnegative . If RNA Parasitic plants that form symplastic connections with their hosts provide a new perspective on cell-to-cell and long-distance trafficking of RNAs. The haustorium creates a union with the host that resembles a graft in many ways, but is established through invasive growth of the parasite and is coordinated with host cells and therefore presents a unique type of junction. Macromolecules including RNAs, proteins, and DNAs, are able to traffic between the plants and the system presents new opportunities for studying RNA movement. Parasitic plants are ultimately pathogens, and the interactions may not be precisely equivalent to the connections between two cells of the same plant, and it is almost certain that parasitic plants have adapted the normal structure and function of PD and RNA trafficking machinery to meet their needs. Unraveling the intricacies of the parasite–host interface is likely to contribute to understanding these phenomena in the same way that studies of viruses hijacking the RNA trafficking system played a major role in elucidating components of PD function . The useAn important feature of the haustorial connection is the ability to connect to diverse host species and this leads to a unique system in which the mobile transcriptome of one species is mixed into that of another. The situation raises intriguing questions about the functions of RNA trafficking in plants. Which RNAs are trafficked, are they targeted specifically, and how do they function at their destination? Are they translated into protein, do they modify gene expression, or are they recycled into raw material for nutrition of the recipient cells? Many of these questions can be addressed using parasitic plants. The ability to readily distinguish host and parasite sequences facilitates identification of mobile transcripts and their movement and fate in the parasite can be tracked using quantitative methods. Next generation sequencing provides the power to reconstruct mobile transcriptomes of hosts from parasite-derived RNA populations.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multiple biomarker testing is necessary to facilitate individualized treatment of lung cancer patients. More than 80% of lung cancers are diagnosed based on very small tumor samples. Often there is not enough tissue for molecular analysis. We compared three minimal invasive sampling methods with respect to RNA quantity for molecular testing.106 small biopsies were prospectively collected by three different methods forceps biopsy, endobronchial ultrasound (EBUS) guided transbronchial needle aspiration (TBNA), and CT-guided core biopsy. Samples were split into two halves. One part was formalin fixed and paraffin embedded for standard pathological evaluation. The other part was put in RNAlater for immediate RNA/DNA extraction. If the pathologist confirmed the diagnosis of non-small cell lung cancer(NSCLC), the following molecular markers were tested: EGFR mutation, ERCC1, RRM1 and BRCA1.Overall, RNA-extraction was possible in 101 out of 106 patients (95.3%). We found 49% adenocarcinomas, 38% squamouscarcinomas, and 14% non-otherwise-specified(NOS). The highest RNA yield came from endobronchial ultrasound guided needle aspiration, which was significantly higher than bronchoscopy  and numerically higher than CT-core biopsy . EGFR mutation testing was feasible in 100% of evaluable patients and its incidence was 40.8%, 7.9% and 14.3% in adenocarcinomas, squamouscarcinomas and NSCLC NOS subgroup respectively. There was no difference in the feasibility of molecular testing between the three sampling methods with feasibility rates for ERCC1, RRM1 and BRCA1 of 91%, 87% and 81% respectively.All three methods can provide sufficient tumor material for multiple biomarkers testing from routinely obtained small biopsies in lung cancer patients. In our study EBUS guided needle aspiration provided the highest amount of tumor RNA compared to bronchoscopy or CT guided core biopsy. Thus EBUS should be considered as an acceptable option for tissue acquisition for molecular testing. Lung cancer is the leading cause of cancer related mortality worldwide with >1.3 million estimated deaths in 2008 However, more then 80% of patients are diagnosed on the basis of very small biopsies or cytology samples The main sources for tissue in advanced lung cancers are bronchoscopic forceps biopsies, CT-guided core biopsies and EBUS-TBNA. The aim of this study was to compare these three sampling methods with respect to the yield of extractable RNA for molecular testing in routinely performed diagnostic procedures of lung cancer patients. To avoid various degrees of RNA degradation by the process of fixation and paraffin embedding, we used a method for tissue banking of diagnostic lung cancer biopsies recently reported by Lawson et al. This study was conducted at Shanghai Pulmonary hospital, Tongji University Shanghai, China in a bilateral cooperation project with Medical Center Mannheim of Heidelberg University. We prospectively screened all patients that were suspicious for lung cancer due to clinical and radiological evidence. Depending on tumor localization, the responsible physician performed bronchoscopy, CT-guided core biopsy or EBUS-TBNA for histological or cytological confirmation of lung cancer.From October 2010 to September 2011, 106 patients who have been diagnosed as NSCLC were considered eligible and enrolled in this study. Five patients were assessed as not evaluable because of insufficient material. All patients with confirmed NSCLC from histological or cytological samples by experienced pathologists were included into this analysis. The histologic diagnosis was based on the World Health Organization classification Biopsies were taken either by endobronchial biopsy forceps  via fiberoptic bronchoscope (Olympus BF-6C260), by endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA)  or by computed tomography-guided needle core biopsy (16–20-gauge Quick-Core Biopsy Needle). Which of these three methods was used for tissue sampling was based on the location of the primary tumor and mediastinal or hilar lymph node status. Once the operator had obtained adequate tissue, biopsies were split into two equivalent parts. Half of the biopsy was sent to pathology. The other half of the biopsy was immediately preserved in RNAlater, which means that samples were placed in an Rnase free Eppendorf tube containing 500 ul of RNAlater (at least ∼10 times the sample volume), and stored at 4°C. If the pathologist confirmed the first half of the sample as NSCLC, then the stored samples were taken out to extract RNA and DNA. In short, the samples were taken out by forceps in RNAlater and cut into pieces on a clean surface. Then, they were transferred into tubes and were processed as described in the handbook. All extracted RNA was quantified. If no diagnosis of malignancy was obtained from the diagnostic biopsies, the study biopsies were removed. Thus, only samples with confirmed NSCLC were stored for further analysis.We used the Allprep DNA/RNA mini kit  to extract and purify the RNA and DNA simultaneously. It is a technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. RT-PCR even needs less RNA to run the gene expression analysis. In our study, we performed the RNA quantification and quality analysis with Nanodrop-2000. RNA and DNA were stored at −80°C for future biomarker analysis.2, 2 ul of 10×reverse transcriptase buffer, 2 ul of 10 mmol/L dNTP, 0.5 ul of RNAse inhibitor, 0. 5 Ug of Oligo (dT) 15, 15 U of AMV reverse transcriptase, filled up to a total volume of 20 uL. Reverse transcription reaction was undergone in a temperature of 42°C for 15 minutes. Then, relative cDNA quantitation for ERCC1, RRM1, and BRCA1 was determined using a fluorescent, real-time detection method  with an internal reference gene (β-actin) as control.All samples were analyzed for mRNA levels of ERCC1, RRM1, and BRCA1. For cDNA synthesis, 2 ul of RNA was preserved in 70°C for 10 min, then added 4 ul of 25 mmol/L MgClAmplification was carried out in a total volume of 25 µl containing 0.25 µmol/L of each primer, 0.02 mmol/L dNTPs and 1 mmol/L MgCl2, 1.25 U Taq polymerase and 5×PCR buffer. The PCR program initiated with 1 min denaturation at 95°C. The DNA was amplified by one cycle of 95°C for 5 s and 50 cycles of 92°C for 40 s, followed by elongation at 60°C for 40 s. The details were described in our previous publications A method for rapid detection of EGFR mutation types in 80 resected NSCLC tissues with PCR combined with Taqman probes was previously reported and described in detail there Standard descriptive statistical analysis for the data was performed. Smoking status was reevaluted into never smokers, and smokers. Tumor stage was classified into advanced (IIIB and IV) and not advanced (IA–IIIA) stage. Testing of groups was done with the two-sided Wilcoxon rank-sum test and in case of multiple testing the Bonferroni-Holms correction for the total significance level was used. Significance level was set to α = 0.05. For descriptive statistical analysis StatXact-9 of Cytel Studio, Version 9.0.0, Cytel Inc., Cambridge, MA, USA and for the tests the SAS software 9.2 (TS2M3) by the SAS Institute Inc., Cary, NC, USA were used.A total of 106 patients with confirmed NSCLC were enrolled into this study, 101 samples were suitable for RNA extraction. Patient characteristics including age, gender, smoking status and tumor related data such as stage, histology and RNA quantity measured in ng/Ul are shown in For all evaluable tumor samples (n = 101), the RNA content was measured with a mean of 24.99 ng/Ul, ranging from 0.01 to 209.20 with a standard deviation of 34.52 . The RNAIn 101 evaluable samples, we detected 25 samples with EGFR mutation , 20 out Results of the three measured biomarkers ERCC1, RRM1 and BRCA1 mRNA Quantity are shown in In the present study we prospectively collected small biopsy samples from a relatively large cohort of untreated patients with newly diagnosed NSCLC to immediately extract RNA and DNA for molecular analysis. Our results demonstrate that all three minimal-invasive methods for tissue sampling - CT-core biopsy, bronchoscopy, EBUS - can provide sufficient material for additional biomarker testing in most cases. It has been reported before, that testing for single molecular markers such as EGFR-mutation from very small tissue samples obtained by EBUS is feasible As an objective measure method for the suitability of sampling, we defined the amount of tumor RNA extractable for biomarker analysis. In our study we adopted the method described by Lawson et al. We found 24.8% of EGFR mutations in our enrolled Asian population, the frequency of EGFR mutations seemed a little lower then expected The main goal of the study was to compare the different techniques used for tissue sampling. Interestingly, EBUS was the best method for securing high amounts of tumor RNA. The content of tumor RNA obtained by EBUS-TBNA was significantly higher compared to bronchoscopy . These findings may be explicable by the known differences of the used methods: bronchoscopic forceps biopsies are taken from tumor periphery. Distinction between necrosis, inflammatory mucosa and vital tumor is not easy through bronchoscopy. Moreover, repeated biopsies may result in bleeding, which often leads to preliminary discontinuation of the procedure without having achieved the optimal tissue yield. CT guided core biopsy may cause pneumothorax, so the procedure will mostly be done only one time to reduce risk of complication and thus only limited amount of tissue is available. EBUS-TBNA on the other hand is a very safe method with a complication rate near to zero In summary, our study confirms that all three different minimal invasive techniques can provide sufficient material for molecular analysis in most of the patients. After testing for the four different markers, there was still RNA left for further analysis of more markers. EBUS-TBNA is a very safe method with almost no risk of complication and has achieved the highest yield of tumor RNA in our study. Therefore it should be an acceptable option for tissue sampling in the era of personalized oncology.


Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides  were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions.Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a K The coding region determinant-binding protein (CRD-BP), also commonly known as insulin-like growth factor II mRNA-binding protein 1 (IMP1), is a member of the conserved VICKZ family of RNA-binding proteins that are characterized by the presence of two N-terminal RNA-recognition motifs (RRMs) followed by four C-terminal KH [hnRNP (heterogenous nuclear ribonucleoprotein) K-homology] domains Drosophila melanogaster, Xenopus laevis and mammalian neuronal cells have provided evidence for the role of IMP1 in nervous system development Mice deficient in IMP1 developed dwarfism and exhibited severe impairment in intestinal phenotype at early birth CRD-BP/IMP1 in human cancers has been extensively studied. While CRD-BP is virtually absent or undetectable in normal adult tissues, it is over-expressed in many types of human cancers including cancers of the breast βTrCP1 mRNA and over-expression of CRD-BP leads to stabilized βTrCP1 mRNA, and elevated βTrCP1 protein levels, resulting in suppression of apoptosis in colorectal cancer cells GLI1 mRNA leading to elevated GLI1 protein, transcriptional activation, and proliferation of colorectal cancer cells myc mRNA K-Ras mRNA and its overexpression led to increases in c-Myc and K-Ras expression as well as colon cancer cell proliferation MDR1 mRNA MDR1 mRNA from degradation is the observation of sensitization of drug-resistant cells to chemotherapeutic drugs upon down-regulation of IMP1 and MDR1 expression The exact mechanism whereby CRD-BP regulates tumour growth and metastasis is still unclear although cumulative evidence suggests its role at the level of post-transcriptional regulation, particularly in its ability to stabilize oncogenic mRNAs. For instance, CRD-BP binds to the coding region of On the contrary to its oncogenic role, there is also evidence of a potential tumour suppressive role for CRD-BP under certain circumstances. The loss of CRD-BP has been shown to lead to the induction of leukemia cell proliferation 2 or 75 cm2 tissue culture flasks and harvested by 0.25% trypsin/0.02% EDTA treatment when they were in logarithmic phase of growth for various experiments.HeLa cervical cancer cells purchased from the American Type Culture Collection  were maintained in MEM  supplemented with 10% fetal bovine serum in a humidified incubator at 37°C supplied with 5% carbon dioxide. Cells were routinely maintained in 25 cmO-methyl derivatives with a phosphodiester backbone. in vitro. For experiments to determine mRNA expression using quantitative real-time PCR, cells were plated onto 6-well plates in a total volume of 2 mL at a density of 5×104 cells/mL. After 24 hours, the cell media were replaced with fresh MEM containing 10% fetal bovine serum and Lipofectamine/oligonucleotide mixture. A similar transfection protocol was applied to the cells after a further 24-hour period of incubation. Forty-eight hours after the first transfection, the total RNA extracted from cells was subjected to quantitative real-time PCR. For Western blot experiments, cells were seeded onto 100 mm dishes in a total volume of 10 mL at a density of 1×104 cells/mL. Cells were transfected at 24 and 48 hours after plating with the oligonucleotides. For RNA immuno-precipitation experiments, the cells were plated onto 100 mm dishes in a total volume of 10 mL at a density of 50×104 cells/mL. In some experiments, cells were then transfected with 10 μg of pcDNA-FLAG or pcDNA-FLAG-CRD-BP 24 hours after plating. Cells were then transfected with oligonucleotides 48 hours after plating. Seventy-two hours after plating, the cells were lysed and subjected to RNA immuno-precipitation as described below. The pcDNA-FLAG-CRD-BP plasmid containing the entire coding region plus parts of 3′untranslated region of mouse CRD-BP was a gift from Dr. Jeffrey Ross, University of Wisconsin.Transient transfection of oligonucleotides was carried out using Lipofectamine 2000 reagent (Life Technologies) as according to the manufacturer’s instructions. As a negative control, the Scrambled Negative (SN) oligonucleotide (IDT Inc.) was used in addition to DD1 and DD10 oligonucleotides, which had no or little effect on the CRD-BP-CD44 RNA interaction Escherichia coli BL21(DE3) cells using a 1 mL bed volume of nickel-NTA (QIAGEN) column under denaturing conditions. Proteins eluted from the column at either pH 5.4 or 4.5 were subjected to a series of dialysis steps (3 hours at each step) pH 5/6 M urea, pH 5.5/4 M urea, pH 6/2 M urea, pH 6.7/1M urea and pH 7.4/0 M urea in a buffer containing 200 mM KCl, 1 mM EDTA, 10% (v/v) glycerol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 0.01% (v/v) Triton X-100, 20 mM triethanolamine The plasmid pET28b(+)-CRD-BP which contains the entire coding region of mouse CRD-BP cDNA was a generous gift from Dr. Jeffrey Ross, University of Wisconsin. The mouse CRD-BP cDNA in this plasmid is flanked with the FLAG tag epitope at its N-terminus and a 6xHis-tag at its C-terminus. Recombinant CRD-BP was purified from myc-1705-1886 was used to amplify the DNA template, which was used to synthesize internally radiolabeled 182 nts c-myc CRD RNA in-vitro transcription by T7 RNA polymerase. Oneμg of DNA template was incubated for 1 hour at 37°C in a 20-μl reaction containing 1 x transcription buffer , 10 mM dithiothreitol, 1 unit RNasin (Promega), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 12.5 μM UTP, 1.5 units T7 RNA polymerase , and 40 μCi [α-32P] UTP (3000 Ci/mmol). Following incubation, 3 units of RNase-free DNase I (Promega) were added and the reaction was further incubated for 10 minutes at 37°C. Upon addition of 10 μl Stopping dye , the entire sample was electrophoresed on a 6% polyacrylamide/7 M urea gel and the band containing internally-radiolabeled RNA was excised and eluted with elution buffer  at 45°C for 6 hours. The purified, radiolabeled RNA was then phenol/chloroform extracted followed by ethanol precipitation. Specific activity of the RNA was then determined by scintillation counting.The previously constructed plasmid pUC19-CRD32P] RNA sample was heated to 50°C for 5 minutes and cooled to room temperature before adding the EMSA binding buffer and the appropriate amount of purified recombinant CRD-BP to a final volume of 20-μL. Reactions were incubated at 30°C for 10 minutes and transferred to ice for 5 minutes. The reaction was again incubated at 30°C for 10 minutes and transferred to ice for an additional 5 min. A total of 2 μL EMSA loading dye  was added to each reaction and 15 μL of the EMSA reaction was loaded onto an 8% native polyacrylamide gel and resolved at 25 mA for 90 minutes. Following electrophoresis, the gel was exposed overnight at –80°C and subjected to autoradiography using the Cyclone PhosphorImager and Optiquant Software.The electrophoretic mobility shift assay (EMSA) binding buffer  d) for the CRD-BP-CD44 RNA (nucleotides 2862–3055) interaction was determined using the Hill equation. The saturation binding data was analyzed by densitometry of the autoradiograph using the Cyclone Storage Phosphor-System and Optiquant software. For each reaction the total activity in each lane was determined; this involved summing the total activity in bound complexes with the total activity present in the unbound fraction. The percentage of bound RNA and the protein concentration (nM) were inserted into the Hill equation and the results were expressed graphically.EMSA saturation binding experiments were carried out as described above and the dissociation constant  and CRD-BP for 10 minutes at 30°C. Following the pre-incubation, 40 nM radiolabeled probe was added to the reaction. This was followed by the standard EMSA protocol as described above. The molar excess concentrations of oligonucleotides over the radiolabeled RNA probe are shown in each figure.myc mRNA levels were examined by quantitative real-time PCR (qPCR). The first strand cDNA synthesis was performed using iScript cDNA Synthesis kit  on 1 μg of total RNA, and the qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) on an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The PCR primers synthesized by IDT Inc. were: CD44 forward primer, 5′-CAT CAG TCA CAG ACC TGC CCA ATG C-3′, and CD44 reverse primer, 5′-ATG TAA CCT CCT GAA GTG CTG CTC C-3′; APE1 forward primer, 5′-TGG AAT GTG GAT GGG CTT CGA GCC-3′, and APE1 reverse primer, 5′-AAG GAG CTG ACC AGT ATT GAT GA-3′; c-myc forward primer, 5′-ACG AAA CTT TGC CCA TAG CA-3′, and c-myc reverse primer, 5′ GCA AGG AGA GCC TTT CAG AG-3′; β-actin forward primer, 5′-TTG CCG ACA GGA TGC AGA AGG A-3′, and β-actin reverse primer, 5′-AGG TGG ACA GCG AGG CCA GGA T-3′. The cycling protocol consisted of 95°C for 3 minutes and 40 cycles of denaturation at 95°C for 10 seconds, and annealing at 52°C for 30 seconds. To confirm amplification specificity, we performed a melting curve analysis at the end of each cycle. Each sample was amplified in triplicate, and the data were analyzed using iQ5 optical system software. Serial dilutions were carried out for each total RNA sample and reverse-transcribed under the above-mentioned conditions for each primer set to ensure amplification with efficiencies near 100%. CT values for target genes  and reference gene (β-actin) were then used in the comparative CT method or commonly known as the 2−ΔΔCT method Total RNA was extracted from cells using the AMBION’s MirVana kit (Life Technologies) as according to the manufacturer’s instructions. CD44, β–actin, APE1, and c-th wash with TCL buffer containing urea, 50% of the agarose beads were collected and spun down at 3000xg for 1 minute followed by resuspension in 16 μL of ddH2O for Western blot analysis. The remaining 50% of the agarose beads were subjected to a final wash followed by resuspension in 100 μL TCL buffer containing 0.3 mg/mL proteinase K and 0.1% SDS and incubated at 50°C for 30 minutes. Following incubation, RNAs physically associated with the resin were extracted by the phenol-chloroform-isoamyl alcohol method and quantified using a Nanodrop Spectrometer . One μg of RNA from each sample group was treated with DNase  before cDNA synthesis and qPCR as described above.HeLa cells transfected with pcDNA-FLAG or pcDNA-FLAG-CRD-BP in a 100 mm dish were lysed with 1 mL Total Cell Lysis (TCL) buffer  supplemented with 1 mM vanadyl ribonucleoside, 0.5 mM DTT, 0.05 units RNasin, and protease inhibitor tablet . After 5 minutes on ice, lysed cells were aspirated using a 26 gauge needle five times to break the nuclei. After a 30-minute incubation on ice, the cell lysate was spun down at 14,000 rpm for 10 minutes. The collected supernatant was subjected to pre-clearing by incubating with 50 μL equilibrated-protein-G agarose beads (50% slurry) at 4°C for at least 1 hour. The resin was spun down at 3000xg for 1 minute and the lysate was collected. The pre-clearing step was repeated once after which the pre-cleared lysate was added to 5 μL anti-FLAG antibody  and mixed overnight at 4°C. Protein G-agarose beads were then added to the antibody-lysate mixture and mixed for 4 hours at 4°C to capture the anti-FLAG antibody. The agarose beads were then washed four times with TCL buffer followed by five washes with TCL buffer containing 1 M urea. Following the 4) were utilized.The protocol for immuno-precipitating endogenous CRD-BP-RNA complex was the same as that described above for immuno-precipitation of FLAG-CRD-BP-RNA complex, except that protein A agarose beads and polyclonal IMP1 antibody , IMP1 polyclonal antibody , and β–actin  antibody. PageRuler Plus Prestained Protein Ladder  was used to approximate the molecular weight of FLAG-CRD-BP, endogenous CRD-BP, and β–actin bands.in-vitro transcribed RNA fragments that cover the entire 3′UTR of the CD44 transcript, a previous study has identified five high affinity RNA binding sites for recombinant IMP1 in-vitro transcription corresponding to the highest affinity sites  and to the lowest affinity binding sites . The four 32P-internally labeled RNA fragments were assessed for binding to four concentrations of purified recombinant mouse CRD-BP ranging from 269 nM to 1885 nM. At the lowest concentration of CRD-BP, 14.1% and 11.5% of Fragment 4 and 8 were bound to the protein  while up to a 20-fold excess of β–globin RNA (lanes 9 and 10) and CD44 RNA corresponding to position 4236–4566 (lanes 7 and 8) have no effect on 32P-labeled Fragment 4 binding to CRD-BP. This confirms that the interaction between Fragment 4 and CRD-BP is specific.We further characterized the interaction between CRD-BP and Fragment 4, which corresponds to position 2862-3055 of the CD44 transcript. We first performed EMSA competition assays to determine if the interaction between Fragment 4 and CRD-BP is specific. 32P-labeled Fragment 4 to better understand the affinity of the interaction between CD44 RNA and CRD-BP. The saturation-binding experiments were done using three different batches of recombinant CRD-BP obtained from replicate purifications. A representative result for the binding of CRD-BP to CD44 32P-labeled Fragment 4 is shown in d) and Hill coefficient (n) were estimated to be 645 nM and 1.7 nM respectively. The Hill coefficient suggests that two molecules of CRD-BP may bind to one molecule of CD44 Fragment 4. Interestingly, similar binding characteristics (affinity and binding stoichiometry) of CRD-BP to a specific coding region of c-myc and MDR1 RNAs have been previously observed We performed saturation-binding experiments using in-vitro transcription  of all nine small molecule antibiotics is shown in 50 of 3.4 μM and paramomycin, the second most potent inhibitor has an IC50 of 58.8 μM. Interestingly, the IC50 value of neomycin for inhibition of the CRD-BP-CD44 RNA interaction is similar to its IC50 values in inhibiting other RNA-protein interactions such as the HIV Tat protein-TAR RNA interaction 50 values of 231.8 and 491 μM respectively. Puromycin, erythromycin, tobramycin, lincomycin and hygromycin were virtually ineffective in inhibiting CRD-BP-CD44 RNA interaction with IC50 values greater than 1000 μM. We next determined whether neomycin can affect the CRD-BP-CD44 mRNA interaction in cells. We first assessed whether HeLa cells can tolerate treatment with small molecules antibiotics, including neomycin, lincomycin and paromomycin, up to 1000 μM. Using light microscopy, we did not see any obvious morphological abnormalities exerted by the small molecule antibiotics even at 1000 μM (unpublished observation). We then treated cells over-expressing the FLAG-CRD-BP with 750 μM neomycin and assessed the level of mRNAs physically associated with FLAG-CRD-BP. In addition to non-treated cells being used as a negative control, we also treated cells with 750 μM of lincomycin, which has no inhibitory effect on CRD-BP binding to CD44 RNA in-vitro , with an affinity similar to other known binding partners such as c-myc and MDR1 RNAs d at around 600 nM. It is also worth noting that a second binding complex could be detected in all EMSAs performed with all CD44 RNA fragments tested. Interestingly, c-myc and MDR1 mRNAs all displayed a similar binding pattern in EMSAs Igf-II and H19 RNA by a sequential, cooperative mechanism The binding affinity of CRD-BP was strongest towards the 194 nts CD44 Fragment 4 RNA  to P4 (nts 2862–2980) , suggestBased on analogy to ZBP-1, the CRD-BP C-terminal KH domains are implicated in RNA binding rather than the N-terminal RNA recognition motifs in-vitro gel shift assay, we tested ten antisense oligonucleotides, which collectively cover the entire CD44 Fragment 4, for the ability to inhibit CRD-BP binding to 32P-labeled CD44 Fragment 4. Only three of these antisense oligonucleotides, namely DD4, DD7 and DD10, were effective as competitors to occlude CRD-BP binding  with four different [32P] CD44 RNA fragments (40 nM), in the absence  or presence of various concentrations of recombinant CRD-BP . The unbound and bound radiolabeled RNAs are indicated. The lower panel shows the positions of the individual RNA fragments  corresponding to the 3′UTR of CD44 mRNA (NCBI accession number: NM_000610).(TIF)Click here for additional data file.


There are errors in Figure S1Heterochiral D/L-RNA-duplex showing either right- or left-handedness. The duplex shown consists of the short sequences D-(5′-CGCCA-3′) and L-(5′-UGGCG-3′) and the base moieties are paired in a canonical Watson-Crick manner. The D-RNA strands are always shown in red, whereas the L-RNA strands are shown in blue. Interestingly, due to the heterochirality of the base pairs, D/L-duplexes can show either right- (A) or left-handedness (B). A. The D/L-RNA duplex shown in its right-handed, anti-parallel conformation. The D-RNA strand (red) is in a natural A-RNA form configuration, whereas the nucleotides of the mirror-image L-RNA strand (blue) show ribose rings, which are rotated about the N-glycosidic bonds, which connect the bases with their ribose rings. This arrangement of the nucleotides leads to a smaller minor groove and a larger major groove of the duplex. One can say that the D-RNA strand has forced its natural (right-)handedness upon the L-RNA strand. Only for comparison reasons: The transparent red D-RNA strand shown arranged together with the opaque red D-RNA strand would form a natural, right-handed D/D-RNA duplex in A-RNA form. The atom positions of the base moieties of the natural D/D-duplex and that of the D/L-RNA duplex are identical. B. The same D/L-RNA duplex, but now shown in its left-handed, anti-parallel conformation. The L-RNA strand (blue) is in its preferred left-handed A-form configuration, whereas the nucleotides of the natural D-RNA strand (red) show ribose rings which are rotated about the N-glycosidic bonds connecting their bases. In this case, one can say that the L-RNA strand, for which the natural handedness is left-handed, has forced its handedness upon the D-RNA strand. For comparison reasons: The transparent blue L-RNA-strand shown arranged together with the opaque blue L-RNA strand would form a conventional, left-handed L/L-RNA duplex.(TIF)Click here for additional data file.Figure S2The detailed view of ribonucleotide conformations in homochiral and heterochiral complexes. The natural nucleotides are shown in red, whereas the mirror-image nucleotides are shown in blue: A. Comparison between the natural D/D-base pair (above) and the right-handed conformation of the D/L-base pair (below). B. Superimposition of the D/D- with the D/L-base pair. C. Diagonal side-view of the D/D-base pair superimposed with the D/L-base pair. As one can see, the D-ribose ring and the L-ribose ring show a common symmetry plane as soon as the necessary ribose ring rotation has taken place.(TIF)Click here for additional data file.


Drosophila melanogaster germline, the piRNA pathway silences retrotransposons as well as other transcribed repetitive elements. Suffix is an unusual short retroelement that was identified both as an actively transcribed repetitive element and also as an element at the 3′ ends of the Drosophila non-LTR F element. The copies of suffix that are F element-independent are far more actively transcribed than their counterparts on the F element. We studied the patterns of small RNAs targeting both strands of suffix in Drosophila ovaries using an RNase protection assay and the analysis of the corresponding RNA sequences from the libraries of total small RNAs. Our results indicate that suffix sense and antisense transcripts are targeted mainly by 23–29 nucleotides in length piRNAs and also by 21 nucleotides in length siRNAs. Suffix sense transcripts actively form longer RNA species, corresponding either to partial digestion products of the RNAi and Piwi pathways or to another RNA silencing mechanism. Both sense and antisense suffix transcripts accumulated in the ovaries of homozygous spn-E, piwi and aub mutants. These results provide evidence that suffix sense and antisense transcripts in the germ line and soma are targeted by both RNAi and Piwi pathways and that a Dicer-independent pathway of biogenesis of siRNAs could exist in Drosophila cells.In the  Drosophila. The first is RNA interference (RNAi), which acts via 21-nucleotide (nt)-long siRNAs that originate from endogeneous long double-stranded RNAs (dsRNAs) and silence mRNAs from retrotransposable elements and host genes There are three distinct RNA-silencing pathways in Drosophila, miRNAs that are partially complementary to mRNAs are generated by Dicer-1, which acts with a dsRNA-binding protein partner termed Loquacious Drosophila requires members of the Piwi subfamily of Argonaute proteins, including Piwi, Aubergine (Aub) and Ago3 Drosophila ovary was described Normal accumulation of somatic endogeneous siRNAs that are complementary to mRNAs requires both Dicer-2, an endoribonuclease that generates siRNAs from long dsRNA, and the RNAi effector protein Ago2 Drosophila mobile elements found in heterochromatin. Such clusters were originally detected many years ago using molecular techniques, but their role was not well understood flamenco, an element found in heterochromatin on the X chromosome, in the transposition regulation of several mobile elements D. melanogaster genome revealed that a small number of clusters of mobile elements or their fragments were trigger loci that produced piRNAs in the germline that repressed many retrotransposons flamenco is the source of piRNAs that target several types of retrotransposons and that flamenco-derived piRNAs almost exclusively occupy Piwi complexes Recently, new research on piRNAs has drawn fresh attention to the clusters of Drosophila that endogeneous siRNAs derived from transposons are generated in somatic cells, while transposon transcripts are cleaved by the Piwi pathway in the germline Drosophila are targeting both protein-coding genes and mobile elements in both gonadal and somatic tissues Suffix is an unusual short retroelement in that there are separate conserved copies of the element, as well as divergent copies, in the 3′ untranslated regions of three genes Suffix has also been identified in the opposite polarity on the 3′ end of the Drosophila non-LTR F element, where it forms the 8th conserved domain of a reverse transcriptase suffix, together with fragments of other retrotransposons, have also been detected in genomic DNA suffix inside microsatellite regions consisting of (CAACA)n repeats suffix have been detected at all stages of Drosophila development, with both suffix-specific siRNAs and longer piRNAs detected in the ovaries Suffix-specific RNAi leads to silencing of the relative LINE (long interspersed nuclear element) F element, suggesting that SINE-specific RNAi could downregulate genes with SINE stretches in their 5′ or 3′ non-coding regions (a phenomenon known as concerted silencing) It was demonstrated recently in suffix sense and antisense transcripts in the ovaries of wild-type flies and in a number of homozygous mutants. We showed that in Drosophila ovaries and somatic cells there is a class of short ago3-dependent piRNAs with the length of 23–29 nt, and that suffix sense and antisense transcripts are silenced by both the RNAi and Piwi pathways. In aub, piwi and spn-E homozygous mutants, both sense and antisense suffix transcripts accumulated at high levels, which independently confirmed that the RNAi and Piwi pathways are involved in cleaving suffix transcripts. Because suffix is actively transcribed into longer transcripts, including mRNAs containing the antisense strand of the element, we surmise that sense suffix-specific 23–28-nt piRNAs may be involved in efficient silencing of suffix-containing mRNAs. In this scenario, suffix could act as a “label” for transcripts that are designated for “concerted silencing,” a suggested mechanism that uses dispersed SINEs sequences in non-coding regions of different mRNAs as targets for RNAi-mediated synchronous silencing of SINE-containing genes The aim of the present study was to study small RNAs targeting Suffix is actively transcribed during all stages of Drosophila development, and both sense and antisense RNA transcripts are found in somatic cells as well as in ovaries and testis suffix transcripts. In preliminary experiments, we optimized conditions for the complete digestion of the gel-purified strand-specific [32P]-labeled suffix transcripts. No protection of the labeled RNA probes was observed in self-annealing or tRNA-annealing experiments  also revealed that the major part of the observed 23–26-nt suffix antisense RNA species belong to a piRNAs class and only a small part corresponds to siRNAs.We observed no partial digestion products that could potentially have formed during dicing of long dsRNAs. The detected antisense 21–26-nt RNAs probably are designated for targeting of 32P]-labeled suffix antisense probe and treating the reactions with RNase, we observed a series of RNA bands in the 19 to 54 nt region of the gel, reflecting a complex pattern of suffix sense small RNAs , suggesting that the larger bands were not the products of incomplete RNase digestion of the probe, but rather corresponded to hybrids with longer suffix sense small RNAs species. Our RNase protection experiments with the [32P]-labeled suffix sense probe independently suggested that in the conditions used the digestion of non-hybridized RNA probes was complete  in suffix dsRNAs and/or during primary piRNA slicing. However, the absence of partially digested Dicer products in these RNA preparations . However, in piwi and spn-E mutants, the increase was much higher: 15- and 12-fold, respectively.RT-PCR analysis also confirmed that suffix sense transcripts. The RT-PCR experiments showed that in the absence of Aub or Piwi, which are critical proteins in the Piwi silencing pathway, suffix sense transcripts accumulate at high levels in the ovaries. The RNase protection experiments detected both 21-nt siRNAs (c 20%) and 23–26-nt piRNAs (c 80%) coming from suffix antisense strand .Analysis of sequences of suffix sense piRNAs in the range 24–29 nt corresponding to the region of suffix used in the RNase protection experiments  are extremely abundant during all stages of Drosophila development Suffix appears in opposite orientation on the 3′ ends of some genes, and the mRNAs of those genes have the suffix antisense sequence on their 3′ ends Suffix-specific RNAi leads to silencing of the relative LINE – F element suffix antisense sequence in genes could also lead to gene silencing. In this manner, a set of genes with a SINE in their 5′ or 3′ ends could also be silenced via concerted silencing suffix sequences can be found in very long polyadenylated transcripts in embryos and imagos Drosophila genome Drosophila melanogaster genome sequence, there are only a small number of separate copies of suffix. Most suffix copies listed in the fly databases are located on the 3′ ends of complete F elements. Full-length copies of this LINE are often surrounded by short fragments of mobile elements in random orientations. However, the separate suffix copies are transcribed far more actively than their counterparts in F elements Both sense and antisense suffix copies inserted in a Drosophila satellite sequence in the form (CAACA)n (work in progress). Between the clustered suffix copies, there are sometimes fragments of other mobile elements inserted in random polarities. This satellite sequence is located in heterochromatin on the right arm of chromosome 2 and on the Y chromosome Drosophila melanogaster genome sequence. A number of other mobile elements in this satellite have been described previously Drosophila 1.688 satellite DNA is transcribed and that its transcription is regulated by RNAi suffix in the (CAACA)n microsatellite regions are transcribed. In theory, they could give rise to the primary piRNAs and long transposon-containing transcripts.We detected separate conserved and clustered diverged Drosophila mutants with increased transposable element mobilization of one particular or different transposable elements were described Years ago, Drosophila requires Dicer-2 ribonuclease and the RNAi effector protein Ago2. We observed that siRNAs were still present in dcr-2 homozygous mutants. Nevertheless, our data also indicated that Dicer-2 was required for formation of suffix-specific siRNAs, because quantities of both suffix sense and antisense siRNAs in the ovaries of the homozygous dcr-2/dcr-2 mutant were reduced as much as 5-fold compared with the wild-type ovaries . It follows that in ovaries suffix transcripts are targeted by both RNAi and Piwi pathways, but piRNAs do most of the work. This conclusion is consistent with the data obtained by the RNase protection assay  has a damaged Drosophila spindle-class gene that affects all known RNA-silencing pathways suffix sense small RNAs in the homozygous mael mutant, and the mutation had no effect on the siRNA content.During the last few years, data related to the separation of small RNAs have usually been illustrated with photographs that show the rather narrow region of the gels in which siRNAs, miRNAs and piRNAs are separated. It seems likely that after Zamore et al. eparated Figure 2The data related to the longer RNAs supports the hypothesis that there are unknown RNA-regulation pathways that act on longer RNA molecules. Nevertheless, a more detailed analysis of longer RNAs is needed before final conclusions can be drawn about whether they are candidates for a new classes of small RNAs involved in RNA-silencing mechanisms.Suffix corresponds to the 3′ end of the Drosophila LINE – F element. The suffix region on the 3′ end of the F element is a hot spot in the production of transposable element-specific piRNAs suffix is more actively transcribed than its cognate LINE Drosophila ovaries, murine testes, and Xenopus eggs and that their biogenesis depends on primary piRNA components but not on ping-pong components Drosophila traffic jam gene also produce sense piRNAs nos 3′ untranslated region spindle-E −/− flies were obtained by crossing 1 st1 spn-E1 e1 ca1ru/TM3, 1 esSb and 1 st1 spn-Ehls3987 e1 ca1ru/TM3, 1 esSb mutants, which have a point mutation in the helicase domain of Spn-E and in the P-element insertion into spn-E, respectively aubergine −/− flies were obtained by crossing QC42/CyOaub and HN/CyOaub mutants piwi −/− flies were obtained by crossing 2piwi and 3piwi mutants, which have a P-ry11 transposon insertion and a PZ insertional mutation, respectively 414/ago2414ago2L811Fsxdcr-2/L811Fsxdcr-2 and r20/maelr20maelsuffix was cloned into the vectors pGEM-1 and pGEM-2 (Promega) as follows. First, this region was amplified by PCR from a cloned suffix copy using the following primers: 5′ cccAAGCTTCACACGCACCCCACC 3′ and 5′ cccgaattCCCTTTCGCCGGAGACGGGAA 3′ . The amplified product was then digested by EcoRI and HindIII and cloned into the pGEM-1 and pGEM-2 vectors.A 77-bp region of Drosophila ovaries, Schneider 2 cultured cells and Drosophila wild-type pupae using Trizol reagent (Invitrogen) according to the manufacturer's instructions. pGEM-1 or pGEM-2 plasmids containing the same 77-bp sequence from suffix were digested completely with HindIII or EcoRI and used as templates for the synthesis of strand-specific [32P]-labeled RNA probes. Next, 91- or 89-nt-long [32P]-labeled RNA , was synthesized in 20-µL reactions containing 1 µg of DNA template, 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 u/µL RNasin, ATP, GTP and CTP (500 mM each), 0.75 µM [α-32P]-UTP , 10 µM unlabeled UTP and 20 u T7 RNA polymerase (Fermentas).Total RNA was isolated from 32P]-labeled RNA probes, for hybridization and for RNase treatment. The [32P]-labeled RNA probes were gel-purified by separation on 52-cm long denaturing 12% polyacrylamide gels, 0.2 mm thick, to isolate the full-length 91- or 89-nt-long RNA species and to remove shorter fragments. About 2–5 µg of total Drosophila RNA were mixed with about 50,000 cpm of labeled RNA in a 20-µL hybridization mixture , heated for 3 min at 100°C and hybridized at 42°C for 16 h. After hybridization, the samples were treated with RNase A/RNase T1 solution according to the manufacturer's instructions. RNase dilutions were determined experimentally in preliminary experiments to ensure complete removal of non-hybridized [32P]-labeled RNA; we used an RNase concentration that slightly affected the protected RNA in order to make sure that non-protected RNA was digested completely. The protected RNA fragments were dissolved in a 5-µL solution containing 90% formamide, 20 mM EDTA and dyes. The probes were separated at 62°C using 12% denaturing polyacrylamide gels that were 0.2 mm thick and 52 cm long. Signals obtained on a phosphorimager were quantified and normalized using rp49 mRNA as an internal reference.The mirVana miRNA detection kit (Ambion) was used for the purification of the -labeled RNAs that make up the suffix sense or antisense strands, respectively. The sequences from the T7 promoter or polylinker are shown in lowercase. The pGEM-1 and pGEM-2 vectors containing short polylinker stretches were used to minimize the non-suffix sequences in the RNA probes.(DOC)Click here for additional data file.Figure S2Control for DNA contamination in the RT-PCR experiments. RT-PCR was performed (37 cycles of amplification) using rp49 primers and RNA isolated from mutant Drosophila lines as templates. About 2 µg of the isolated RNA was treated with DNase using a DNA-free kit (Ambion) and then used for cDNA synthesis. Reverse transcription was performed using a specific primer and M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. For each set of PCR reactions, the cDNA template (RT+) and the same RNA probe without addition of reverse transcriptase (RT−) were used. DNA contaminating the RNA samples was digested to levels below the PCR detection limits in the conditions used. This supports the idea that the data in (EPS)Click here for additional data file.Figure S3Validation of the RNase protection assay using chemically synthesized RNAs. 21, 25 and 27 nt long RNAs corresponding to the sense suffix RNA were phosphorylated by T4 polynucleotide kinase using unlabeled ATP. After annealing of RNAs with 89-nt long [32P]-labeled suffix antisense RNA and treatment with RNases (see SmaI endonuclease. The data demonstrate that the length of protected [32P]-labeled suffix antisense RNA fragments corresponds to the expected after hybridization with the RNAs used.ases see  the RNAs(EPS)Click here for additional data file.Figure S4Drosophila ovaries contain suffix sense 26–29 nt long piRNAs. The gel presented in suffix sense transcripts that might be present at lower levels due to very strong RNase treatments. The bracket marks RNA bands in the 23–29 nt region that correspond to suffix sense piRNAs.(EPS)Click here for additional data file.