Background: The crystal structure of viral proteins is not available for many plant viruses including tomato spotted wilt virus (TSWV).Results: By homology modeling, we mapped the RNA binding sites and discovered a protective feature of TSWV nucleocapsid (N).Conclusion: Homology modeling provided a basis for functional analysis of TSWV N interacting with RNA.Significance: This approach might be applicable for other plant viruses. The nucleocapsid (N) protein of tomato spotted wilt virus (TSWV) plays key roles in assembling genomic RNA into ribonucleoprotein (RNP), which serves as a template for both viral gene transcription and genome replication. However, little is known about the molecular mechanism of how TSWV N interacts with genomic RNA. In this study, we demonstrated that TSWV N protein forms a range of higher ordered oligomers. Analysis of the RNA binding behavior of N protein revealed that no specific oligomer binds to RNA preferentially, instead each type of N oligomer is able to bind RNA. To better characterize the structure and function of N protein interacting with RNA, we constructed homology models of TSWV N and N-RNA complexes. Based on these homology models, we demonstrated that the positively charged and polar amino acids in its predicted surface cleft of TSWV N are critical for RNA binding. Moreover, by N-RNA homology modeling, we found that the RNA component is deeply embedded in the predicted protein cleft; consistently, TSWV N-RNA complexes are relatively resistant to digestion by RNase. Collectively, using homology modeling, we determined the RNA binding sites on N and found a new protective feature for N protein. Our findings also provide novel insights into the molecular details of the interaction of TSWV N with RNA components. Bunyaviridae family of negative-stranded RNA viruses comprises five genera, namely Orthobunyavirus, Hantavirus, Phlebovirus, Nairovirus, and Tospovirus  of TSWV is the main structural protein that assembles the genomic RNAs into ribonucleoprotein complexes (RNPs) . The RNPPhlebovirus) (–Nairovirus)  –16, Crimrovirus) , 18, andra virus , Schmallra virus , Leanyerra virus , and La ra virus , have alus genus , but nonus genus –26. WithIn this study, we developed blue native gel analysis, homology modeling, and other approaches to investigate the TSWV N-N oligomerization and the interaction of TSWV N protein with genomic RNA. We demonstrated that the TSWV N protein forms a range of higher ordered oligomers. Analysis of the RNA binding behavior of N protein using two different approaches revealed that no specific oligomer binds to RNA preferentially, instead each type of N oligomer is able to associate with RNA. To better characterize the N protein in its interaction with genomic RNA, we constructed homology models of TSWV N and N-RNA complexes. Based on these homology models, we mapped the RNA binding sites onto its predicted surface cleft of TSWV N. Moreover, by N-RNA homology modeling we found that the RNA component is deeply embedded in the protein cleft of N; consistently, RNase A treatment assay revealed that TSWV N-RNA complexes are relatively resistant to digestion by RNase. Taken together, using homology modeling we determined the RNA binding sites on the TSWV N protein and obtained new insights into the protection function of N-RNA complexes.JF960235.1) . The DNA amplicon was digested with NdeI and SacI restriction enzymes and cloned into modified bacterial expression vector pET28a (modified to contain with a tobacco etch virus cleavage site) downstream of the His6 tag. The alanine substitution mutations of N protein on Arg60, Lys65/Lys68, Lys81, Arg94/Arg95, Lys183/Tyr184, Lys192/Thr195 were created by PCR-based site-directed mutagenesis based on pET28-N for bacterial expression. The entire coding region of each mutant was confirmed by DNA sequencing. The RNA transcription plasmid pMD19-T7-5′UTR-GFP-3′UTR with the TSWV termini sequence was constructed by cloning the coding region of green fluorescent protein (GFP) between the TSWV S segment 5′- and 3′-untranslated region (UTR) under the control of the T7 promoter, then cloning it into the pMD19 vector .The coding region of the N protein was amplified from the cDNA of TSWV Yunnan isolate (accession 60235.1)  using foEscherichia coli Rosetta (DE3) was transformed with pET28-N and its derivatives. For expressing proteins, a small overnight culture (10 ml) was transferred to a 1-liter culture and allowed to grow at 37 °C until the optical density at 600 nm reached 0.6. Protein expression was then induced with 0.1 mm isopropyl β-d-thiogalactopyranoside overnight at 25 °C. The cells were harvested and resuspended in cold lysis buffer . After pre-treatment with lysozyme for 30 min, cells were lysed by sonication for 20 min on ice. Cell debris was removed by centrifugation at 12,800 × g for 1 h. The supernatant was incubated with 1 ml of nickel-nitrilotriacetic acid resin  at 4 °C for 2 h and then loaded onto a chromatographic column (Bio-Rad). The Ni-NTA resin was washed three times with 50 mm wash buffer . The proteins were eluted with 250 mm imidazole elution buffer . The protein was dialyzed in storage buffer  and stored at −70 °C until use. To purify the free-tag TSWV N protein, we removed the N-terminal His6 tag from the recombinant N protein, which was expressed from pET28a-N by a recombinant His6-tobacco etch virus protease as described by Wu et al. . PEI was added slowly into the lysate with stirring until the final concentration reached 0.1% (w/v). The nucleic acids were then removed by centrifugation at 12,800 × g for 15 min. The supernatant was further purified by Ni-NTA affinity chromatography.To remove the endogenous Total RNA was isolated from the N-RNA complex of the wild type and mutant proteins by adding 2× RNA gel loading buffer containing 95% formamide, which effectively denatures and dissociates RNA from the protein. After boiling at 100 °C for 5 min, the samples were electrophoresed in 1.0% nondenaturing agarose. RNAs were stained with ethidium bromide and visualized using long wavelength UV light. For analysis of the cellular RNA bound to each oligomeric type of N protein, the proteins were recovered from the blue native gel and mixed with RNA loading buffer to release the RNA and analyzed in 1.0% agarose gel.et al.  and incubated for 10 min at room temperature. Protein samples were loaded onto 4–16% discontinuous blue native PAGE gels and separated at 4 °C in a cathode buffer containing 100 mm histidine (adjusted to pH 8.0 using Tris base) and 0.002% Coomassie Brilliant Blue G-250. The gels were fixed and destained in 7.5% acetic acid and 5% ethanol. For isolation of different oligomers from blue native gels, the corresponding band was precisely cut from the native gels immediately after electrophoresis was completed, the gel was minced, and the protein was eluted in phosphate-buffered saline (PBS). For analyzing the oligomeric dynamics of N protein, each recovered oligomeric type of N protein was run in a new 4–16% discontinuous native PAGE gels.The blue native gel electrophoresis analysis was carried out as described by Wu et al. . Brieflym Tris-HCl, pH 7.5. The protein samples were then boiled in SDS-denaturing buffer, and separated in 5–20% SDS-polyacrylamide gradient gels. Proteins were detected by Coomassie Blue staining.The purified N protein (30 μg) was mixed with various concentrations of freshly prepared glutaraldehyde solution, then incubated at 37 °C for 5 min. The reactions were quenched by adding 2 μl of 1 in vitro Transcription kit . RNA transcripts were synthesized in a reaction mixture containing 2.5 mm (each) ATP, CTP, GTP, and UTP. DIG-labeled riboprobes were synthesized in 20-μl reaction volumes containing 1 mm NTP or 1 mm (each) ATP, CTP, and GTP, 0.65 mm UTP, 0.35 mm DIG-11-UTP (Roche Applied Science), 500 ng of linearized template, 20 units of RNase inhibitor, and 40 units of T7 polymerase and incubated at 37 °C for 2 to 3 h. All reaction mixtures were then treated with 1 μl of DNase I for 15 min at 37 °C. The transcripts were purified by phenol-chloroform extraction and ethanol precipitation in the presence of ammonium acetate and then dissolved in RNase-free, double-distilled H2O (ddH2O). The concentrations of RNA transcripts were determined by measuring absorption at A260 with a NanoDrop 1000 spectrophotometer .RNA transcripts were generated using the T7 Riboprobe Protein concentrations were determined using the Bio-Rad protein assay kit. Protein samples were treated with 3× SDS buffer. After boiling for 7 min, the samples were separated by electrophoresis in 10% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The antigens on the membrane were incubated with a monoclonal antibody against TSWV N   and subsThe homology model of TSWV N protein was constructed using the on-line server at I-TASSER , 26. Them) was mixed with increasing concentrations of wild type or mutant N proteins. Reaction mixtures were incubated at room temperature for 10 min and filtered through two layers of membranes under vacuum: the top membrane (nitrocellulose) binds proteins and RNA-protein complexes, and the bottom membrane (nylon which is charged) collects free RNA. Filters were washed with 10 ml of RNA binding buffer. The amount of RNA retained on the filter was detected by digoxigenin-AP Fab fragments (Roche Applied Science) followed by BCIP/NBT visualization. The bound RNA was quantified as described by Wu et al.  with 3 nmol of DIG-11-UTP-labeled RNA probe (5′UTR-GFP-3′UTR). The reaction mixtures were incubated for 10 min at room temperature, then 5 μl of loading buffer  was added. The reaction mixtures were separated by electrophoresis in 1% agarose with 0.5× TBE . The gel was electroblotted onto a nylon membrane . The electrophoretic retardation of the DIG-labeled RNAs was detected by AP-labeled anti-digoxigenin followed with BCIP/NBT staining.In a typical EMSA, increasing amounts of N or mutants proteins were incubated in 10 μl of binding buffer . The reaction mixtures were incubated at room temperature for 10 min. RNase A was then added to the concentrations specified, and the mixtures were further incubated at 37 °C for 30 min. The products of the binding reactions were then separated by electrophoresis in 1% agarose. The gel was electroblotted onto a nylon membrane . The RNA on the membranes was fixed by 0.25 J/cm2 of UV light . The effects of RNase A on the N-RNA complexes or mutant-RNA complexes on the membrane was detected using AP-linked anti-digoxigenin antibody, followed by signal development in ready-for-use BCIP/NBT solution.The DIG-labeled RNA probe with the TSWV termini  was incubated in the presence or absence of 2 μg of TSWV N or non-RNA binding mutant protein in a total reaction volume of 10 μl in binding buffer . Further analysis by Western blot using the monoclonal antibody raised against the TSWV N protein confirmed the identity of the recombinant protein as TSWV N (A).To purify the TSWV N protein for biochemical characterization, we fused the N protein with the N-terminal Hiss TSWV N A.A at 260 to 280 nm (A260/A280) for the purified TSWV N protein was up to 1.7 (data not shown), which indicated that the recombinant TSWV N protein was bound to E. coli endogenous RNA. To substantiate this observation, we denatured the purified N protein and analyzed the released RNA in a 1% agarose gel. We included a recombinant maltose-binding protein expressed in the same strain of E. coli as a control. As shown in Fig. 1B, the N protein was readily detected with RNA in the agarose gel staining with ethidium bromide, whereas the recombinant maltose-binding protein had no detected cellular RNA. These results suggest that the TSWV N protein binds nonspecifically to E. coli cellular RNA. The observation of nonspecific binding of TSWV N protein to RNA is consistent with a previous report that N protein binds to RNA irrespective of sequence as described by Richmond et al. . To further examine the oligomerization state of the N protein, we cross-linked the recombinant N protein by adding increasing amounts of glutaraldehyde. As the concentration of glutaraldehyde increased, an intensified band of dimers, trimers, and higher oligomers appeared , confirming that the N protein was able to form different higher ordered oligomers. Hence, TSWV N was able to form a range of higher ordered oligomers.To investigate the oligomerization state of TSWV N, we used a discontinuous blue native gel electrophoresis analysis, which allows the separation of the protein complex in its native quaternary structure according to its size and shape , 30–33. 30–Fig. 2E. coli RNA nonspecifically, we first removed the E. coli RNA from the N protein by PEI treatment. RNA gel analysis showed that PEI-treated N protein was largely RNA-free (A). To check the oligomeric formation of RNA-free N protein, we determined the PEI-treated protein along with N protein without PEI treatment in the discontinuous blue native gel. PEI-treated N protein formed a similar pattern of higher ordered oligomers compared with those of N protein without treatment (B). It strongly suggests that higher ordered oligomeric formation of the N protein does not depend on the binding of RNA. The bands of higher ordered oligomers formed by PEI-treated protein ran faster than those by the non-treated protein, which may be due to the smaller size of complexes formed by RNA-free N protein. We next carried out an electrophoresis mobility shift assay to analyze the binding behavior of N oligomers in the discontinuous blue native gel. A fixed amount of PEI-treated N protein (15 μg) was incubated with increasing amounts of RNA transcripts with the TSWV termini sequence representing the 5′-UTR and 3′-UTR of the TSWV genomic S fused with GFP at both ends , and the reaction mixtures were analyzed in a 4–16% blue native polyacrylamide gradient gel. As shown in Fig. 3C (upper gel), when 1 to 10 μg of RNA was added, the oligomers of N did not shift in an obvious manner. At 20 to 80 μg of RNA, the monomer, dimer, trimer, tetramer, and pentamer bands all disappear. The disappearance of those bands strongly implies that they bind RNA to form higher ordered protein-RNA complexes. To rule out the possibility that the disappearance of protein bands is due to the contamination of protease in the synthesized RNAs, the same protein-RNA complexes were also analyzed in 10% denatured SDS-PAGE gel. No degradation was found for those protein complexes .Given that N protein formed different types of higher ordered oligomers, we next determined the RNA binding behavior of N protein oligomers. Because N protein binds to RNA-free A. To chereatment B. It strE. coli cellular RNA. The purified N protein was separated by 4–16% discontinuous blue native PAGE. Each type of N oligomers (number 1–4) and a region of the gel that does not contain N protein (number 5) was isolated from the native gel, respectively . The recovered protein was denatured to release the cellular RNA, then separated in a 1% agarose gel. As shown in Fig. 3D (right panel), the monomers, dimers, trimers, and tetramers all contained E. coli cellular RNA, whereas the region of the gel without N protein did not contain RNA, suggesting that each type of N oligomer was able to bind RNA.The above experiments suggest that each type of TSWV N oligomer may be able to bind RNA. To substantiate this observation, we took advantage of the features that TSWV N protein binds to An earlier study reported that both the N amino and carboxyl halves of TSWV N are involved in binding to RNA . HoweverA and B, the homology structure of N protein has four parts: the N-arm (yellow), N-terminal domain (red), C-terminal domain (green), and C-arm (blue). The N-/C-arms comprised sequences from the N and C termini, which may play an important role in mediating the N-N interaction. The N- and C-terminal domains of the N protein forms a core structure. Displaying the surface of the N- and C-terminal domains demonstrated that a protein cleft was formed between the N- and C-terminal domains (E). Superposition of the homology structure of TSWV N onto the crystal structure of Bunyawera virus N (C and D).To better characterize the RNA binding sites and other features of the N protein in its interaction with RNA, we built a three-dimensional homology model of TSWV N protein using an on-line platform for protein structure and function predictions, I-TASSER, based on multiple threading alignments. As shown in  domains E. Superp virus N  showed a virus N , C and Dblue in F), which may be responsible for RNA binding. We then built an 11-nt RNA into the homology model of the N protein. In the homology model of the N-RNA complexes, the RNA fit well into the surface cleft of the N protein. The RNA component is deeply embedded in the protein cleft (E and F), suggesting a possible structure protection for the packaged RNA.Analysis of the electrostatic surface potential of N homology structure showed a charged surface cleft blue in F, which in cleft , E and FTospoviruses in GenBankTM and aligned the sequences. The positive-charged and polar amino acids, which are conserved in the N protein sequence (E and F), were selected for site-directed alanine substitution mutagenesis. We then generated protein mutants R60A, K65A/K68A, K81A, R94A/R95A, K183A/Y184A, and K192A/T195A, respectively. All N alanine substitution mutants were expressed in E. coli, treated with PEI, and purified using Ni-NTA affinity chromatography. The oligomeric formation of alanine-substitution mutant proteins was examined in the 4–16% discontinuous blue native gel. As shown in Fig. 6A, all N alanine-substitution mutants displayed a similar pattern of oligomeric formation compared with the wild-type N protein, suggesting that oligomeric formation by the mutants did not change substantially.The homology model of the TSWV N structure suggested that the charged surface cleft of the N protein may be responsible for RNA binding. To determine the amino acids of N protein that are involved in RNA binding, we obtained nucleocapsid sequences from the 20 sequence  and locasequence , E and FB), suggesting that the RNA binding affinity of both R94A/R95A and K183A/Y184A was significantly reduced. To verify this observation, we further performed a gel mobility assay for these two mutants. A fixed amount of DIG-labeled RNA transcripts (3 nmol) was incubated with increasing concentrations of R94A/R95A and K183A/Y184A mutant protein, respectively. Compared with the wild-type protein, R94A/R95A and K183A/Y184A had significantly less RNA-binding ability in a gel mobility assay (C). These data demonstrated that amino acids Arg94, Arg95, Lys183, and Tyr184 are important for N binding to RNA. When those amino acids are displayed in the three-dimensional structure of N homology model, they mapped onto the surface cleft of N protein (D and E).To examine whether any mutant was defective in RNA binding or reduced in their binding ability, we determined the dissociation constant for each of alanine-substitution mutant using a filter binding assay. A fixed amount of DIG-labeled RNA containing the TSWV termini sequence  was mixed with increasing quantities of wild-type N or mutant proteins. Quantification analysis of the RNA-protein complexes retained on nitrocellulose showed that the dissociation constant for mutants R60A, K65A/K68A, K81A, and K192A/T195A was comparable with the wild-type N protein; however, the dissociation constant for mutants R94A/R95A and K183A/Y184A was greater than that of wild-type N protein B, suggesty assay C. These  protein , D and EA). To ensure that the N protein-RNA interaction, which is directly providing resistance to RNase A, non-RNA binding variants of the N protein, R94A/R95A, and K183A/Y184A, were used for the RNA protection assay, respectively. DIG-labeled riboprobe  was incubated with 2 μg of R94A/R95A or K183A/Y184A mutant protein, then increasing amounts of RNase A were added. As shown in B and C, the addition of 0.005 μg/ml of RNase A completely digested the RNA from mutant-RNA complexes, implying that the mutant that lost the RNA-binding activity was no longer able to protect the RNA from RNase treatment. In summary, these results suggested that the TSWV N protein can protect RNA from degradation by RNase.From the homology model of N-RNA complexes, we observed that the modeled 11-nt RNA component is deeply embedded in the protein cleft. Such a structure may protect the RNA from digestion by RNase. To address whether the TSWV N was indeed able to protect the RNA by the degradation of RNase, DIG-labeled riboprobe  was incubated with 2 μg of PEI-treated N protein to form N-RNA complexes, then increasing amounts of RNase A were added. When 0.005 μg/ml of RNase A was added, the band of N-RNA complexes became broader and shifted a little lower, which may be due to partial digestion by RNA in N-RNA complexes. However, the remaining RNA complexed with the TSWV N protein was still resistant to RNase A. When the concentration of RNase A was increasing, RNA complexed with the N protein was partially resistant to digestion with up to 5 μg/ml of RNase A A. To ensIn this paper, we reported structure and function analysis of TSWV N interacting with RNA using homology modeling. Based on homology models of TSWV N and N-RNA complexes, we mapped RNA binding sites onto its predicted surface cleft of TSWV N. By N-RNA homology modeling we also discover a new protective feature of N-RNA complexes against the degradation of RNase. Together with the results showing that the N protein forms different orders of oligomers and each oligomer binds to RNA, our findings add new insights into the molecular details of the interaction of TSWV N with RNA components.Tospovirus is the only genus that infects plants (C and D). Despite the constructed homology model may not be the true structure of N protein, the modeled structures have provided us a basis that we can use for evaluating the structure and analyzing the biological functions of the N protein. Thus, our study demonstrated an excellent example that homology modeling is a useful approach for functional analysis of plant viral proteins.The homology model of TSWV N we constructed by I-TASSER showed that the N- and C-terminal domains form a charged surface cleft that has the potential to bind RNA. The 11-nt RNA was modeled and fit well in this electropositive-charged groove. Further alanine substitution analysis confirmed that the positively charged and polar amino acids in the surface cleft of the N protein are indeed responsible for RNA binding. Compared with all other mammalian viruses in the family of Bunyaviridae, s plants . Althougs plants , C and Det al. (A). R94A/R95A or K183A/Y184A mutant that lost the RNA-binding activity was no longer able to protect the RNA from RNase treatment (A–C), suggesting that it is the N protein-RNA interaction that is directly providing resistance to RNase A. The observations that the RNA-N complexes were resistant to RNase treatment have also been reported for the Bunyamwera virus (–In the homology model of the N-RNA complexes, the modeled 11-nt RNA component was found to be deeply embedded in the surface cleft of the N protein. Based on this observation, a further RNase A treatment assay revealed that RNA complexed with the TSWV N protein were relatively resistant to the digestion by RNase. Richmond et al.  have preet al. A. R94A/Rreatment , A–C, sura virus , 34, 35.ra virus –22. The et al. (Phlebovirus, also forms tetrameric, pentameric, or hexameric structures (In a yeast two-hybrid study, N proteins were shown to interact with each other in a head-to-tail manner . In the et al.  also sugructures , 15. FroIn conclusion, using homology modeling, we mapped the RNA binding sites onto the predicted surface cleft of the TSWV N protein and gained new insights about the protective function of TSWV N-RNA complexes. The N protein of TSWV has been shown to be a multifunctional protein that is involved in assembly of RNPs , 38, vir


C. elegans. Ketting and colleagues identify PID-1 as a 21U-RNA biogenesis factor and show that PID-1 affects an early step in the processing or transport of 21U precursor transcripts. The authors further show that maternal 21U-RNAs are essential to initiate silencing. This work provides novel insights into piRNA-induced silencing and small RNA biogenesis.The Piwi–piRNA pathway represents a small RNA-based mechanism to silence invading DNA. It is unknown how transcripts are selected for generation of 21U-RNA, the piRNA class in  Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing.The Piwi–piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In  One of the challenges faced by germ cells is the faithful inheritance of their genomes over generations. An important aspect of this process is to ensure that potentially harmful DNA sequences are not expressed and/or do not replicate. Such sequences include transposons and retroviruses and, when left unchecked, create havoc, leading to fertility problems. One of the mechanisms controlling this line of defense is the Piwi–piRNA system .The Piwi–piRNA pathway is a small RNA-based mechanism. At its core, Argonaute proteins from the Piwi subfamily use small RNA cofactors called piRNAs to recognize and silence transcripts derived from repetitive elements. Silencing often involves the amplification of a small RNA population and can occur at both the post-transcriptional and transcriptional levels. Post-transcriptional silencing involves cleavage of targeted mRNA molecules by the RNase H activity of Piwi proteins, while transcriptional silencing likely involves the recognition of a nascent transcript and is accompanied by the acquisition of repressive chromatin marks at the targeted locus . This heCaenorhabditis elegans, the Piwi protein PRG-1 is guided to its targets by piRNAs (named 21U-RNAs in C. elegans)   in a proelegans) . Combineelegans) , 21U-RNAelegans) . In ordend rde-3 . The 22Gnd rde-3 . The finnd rde-3 . Intrigund rde-3 .The least-understood step in this PRG-1-mediated mechanism is how 21U-RNAs are made. In fact, we only know that 21U-RNA genes are marked by the presence of an upstream consensus motif  of one thenn-1 mutant background with EMS and scored for full activation of the 21U sensor . Genome-wide resequencing of retrieved mutant strains identified premature stop mutations in mut-7 and rde-3 . We also identified an interesting allele (xf25) of hrde-1 carrying a nonsynonymous mutation affecting the last amino acid of HRDE-1 (A1032T). Given the important role for the most C-terminal residue of Argonaute proteins in making the binding pocket for the 5′ phosphate of the small RNA cofactor , showing that HRDE-1(A1032T) is nonfunctional. In summary, our screen effectively identifies genuine components of the C. elegans piwi pathway.In order to identify novel genes acting in the PRG-1-mediated silencing pathway, we made use of a strain expressing a GFP-Histone-2B fusion gene that carries a specific 21UR1-RNA recognition site in its 3′ untranslated region (UTR) (21U sensor) . We prevcofactor , we reasxf14. The locus affected by xf14 was named pid-1. To probe the generality of the apparent 21U accumulation defect, we analyzed small RNAs from both wild-type and pid-1(xf14) young adult animals that also carry the 21U sensor through deep sequencing . Both libraries have a very similar size and display similar microRNA (miRNA) profiles , indicating that the libraries can be well compared.Most of the alleles identified in our screen displayed normal levels of 21U-RNAs, as assessed by Northern blotting , except for pid-1(xf14) mutants these small RNAs still display all 22G characteristics, their levels are strongly reduced .As expected, in wild-type animals, we could detect small RNAs mapping to the 21U sensor . These small RNAs display all characteristics of 22G-RNAs . While in pid-1(xf14) mutants (pid-1(xf14) . The loss of 21U-RNAs is slightly less than observed in prg-1(pk2298) mutants .We next checked mature 21U-RNA levels, normalizing to total miRNA counts. We found that, overall, 21U-RNA levels were 10-fold reduced in  mutants  and that mutants , suggestpid-1(xf14) leads to a strong decrease in type 1 as well as type 2 21U-RNAs . This revealed that transcript levels of prg-1 are unaffected by pid-1. In contrast, PRG-1 protein levels were strongly reduced upon mutation of pid-1 . Most likely, this reflects competition for components shared by the exo-RNAi machinery and the prg-1 pathway, such as MUT-7 and RDE-3.Since many PRG-1 pathway components affect RNAi triggered by exogenous dsRNA, we also probed the RNAi sensitivity of pid-1 gene, we first crossed the pid-1(xf14) allele into a henn-1 wild-type background. This showed that the loss of 21U-RNAs in pid-1(xf14) animals is henn-1-independent . Genetic mapping revealed that pid-1 is genetically closely linked to the 21U sensor transgene on chromosome II (data not shown), and genome-wide resequencing revealed three nonsilent mutations in the area where pid-1 was mapped; these three mutations affect F18A1.8, ZK1320.5, and F37H8.3. We then selected recombinants that separated the 21U sensor from these three mutations. Only recombinants that picked up a wild-type copy of F18A1.8 produced 21U-RNAs , suggesting that F18A1.8 is pid-1. To test this, we generated two additional alleles (xf35 and xf36) of F18A1.8 using CRISPR technology and probed the effect of these alleles on the activity of our 21U sensor and on 21U-RNA biogenesis. First, xf35 and xf36 do not complement pid-1(xf14) . Second, pid-1(xf35) was unable to silence a 21U cherry sensor transgene coming from a mut-7(pk204) mutant background (xf35 and xf36 have a similar effect on mature 21U-RNA levels as pid-1(xf14), as revealed by deep sequencing (pid-1(xf14) mutants, PRG-1 levels are strongly reduced in xf35 and xf36 mutant extracts  results in an arginine-to-cysteine change at position 61 (R61C) (pid-1(xf14) mutant extracts, while the xf35 and xf36 alleles do not produce detectable PID-1 protein at all  . This mun at all . Next, w elegans .KREFSHITLASTPFKKR), and the protein may contain a nuclear export signal (LSAGLSL) as well , indicating that the remaining 21U-RNAs in pid-1(xf14) mutants are still 2′-O-methylated. Furthermore, the global requirement of pid-1(xf14) for 21U-RNA accumulation is also observed in the libraries made from oxidized RNA .To further probe at which level PID-1 affects 21U-RNA biosynthesis, we NaIO elegans , and it We also checked whether the step right before methylation is affected by PID-1. This step involves the trimming of the 21U 3′ end, presumably by an exonuclease. To assess this, we profiled the length distribution of those 21U reads from the TAP-treated libraries whose 5′ ends match precisely onto an annotated 5′ end of a mature 21U-RNA, indicating that the 5′ end has been processed (see below). This revealed no differences between the wild-type and mutant 21U-RNAs . We conclude that PID-1 does not affect the 3′ end processing of 21U-RNAs.prg-1(pk2298) small RNA-derived cDNA libraries. This revealed that in the absence of PRG-1, ∼80% of the reads derived from 21U loci correspond to precursor transcripts, starting mostly at position −2 and extending to ∼35 nt in length ;21U sensor males into either wild-type or pid-1 mutant hermaphrodites. As expected, offspring of these males mated with pid-1-defective hermaphrodites show strong transgene activity. Interestingly, however, when mated to wild-type hermaphrodites, these males sire offspring with active transgenes, albeit the fluorescence intensity is notably lower (prg-1(pk2298);21U sensor males mated with wild-type hermaphrodites. Full silencing of the transgene is only observed in the next generation  males into both wild-type and pid-1 mutant hermaphrodites and analyzed transgene expression in the F1. This revealed a very strong maternal effect of 21U-RNAs: The wild-type hermaphrodites produced fully silenced offspring, while the progeny of the pid-1 mutant mothers could not initiate detectable silencing even though they were heterozygous wild type for pid-1. This result highlights at least two properties of the 21U pathway. First, the maternally provided 21U-RNA pool is essential to establish resilencing of a mut-7(pk204)-activated transgene, suggesting that the initiation of silencing by 21U-RNAs happens during embryonic development using maternally provided 21U-RNAs. Second, a mut-7-activated transgene coming in via the sperm is easier to resilence by 21U-RNAs than a pid-1 or a prg-1-activated transgene. We conclude that some silencing information must be present in mut-7-derived sperm but is lacking in prg-1-defective sperm and that this information can be efficiently used by the maternal 21U-RNA pool.Next, we crossed an active 21U sensor from pid-1, that plays an important role during 21U-RNA biogenesis in C. elegans. PID-1 is a mostly cytoplasmic protein that likely acts during the processing of 21U-RNAs downstream from their transcription. The mature 21U-RNAs that are still made in pid-1 mutants are indistinguishable from wild-type 21U-RNAs, and, relative to these mature 21U-RNAs, 21U-RNA precursors accumulate upon loss of PID-1. Furthermore, the structure of 21U precursor transcripts is not affected by PID-1. Given these results as well as the potential nuclear import and export signals on PID-1, we suggest that PID-1 may somehow guide 21U precursors to a site where they are further processed into mature 21U-RNAs. We note that in other species, like mice or Drosophila, PID-1 orthologs cannot be identified through BLAST analysis. This may be related to the fact that, overall, the 21U-RNA pathway in C. elegans deviates significantly from piRNA pathways in other animals. However, given the still poor characterization of piRNA biogenesis in general, proteins with similar functions but with diverged sequences may operate in mammalian or fly piRNA biogenesis.We identified a novel gene, pid-1 as well as prg-1 partially inhibit RNAi triggered by exogenous dsRNA. Much like what is described for other RNAi-hypersensitive mutants, we attribute this to the fact that the prg-1 pathway included proteins shared with the exo-RNAi pathway  with CRISPR/Cas9 as previously described . The seqThe screen for Pid mutants was performed using a Zeiss M2Bio dissecting microscope. Fluorescence images were taken on a Leica DM6000 B upright microscope with 400× amplification. Silencing onset of active transgenes was analyzed using a Leica M165 FC stereo microscope with a 1× objective (120× amplification) or a 4× objective (480× amplification).E. coli expressing dsRNA against pos-1  was used in a 1:200 dilution on Western blots.g to remove debris. The supernatant (crude extract) was centrifuged at 2000g to separate the nuclear fraction from the cytoplasmic fraction. Nuclear fractions were washed twice with extract buffer, whereas the cytoplasmic fraction was centrifuged at 21,000g to remove any remaining debris.Synchronized adult worms were washed with extract buffer  and resuspended to a final volume of 4 mL. The suspension was then dripped into liquid nitrogen, and the frozen pellets were ground to a fine powder. The powder was transferred to a glass douncer, thawed on ice, and sheared . The extracts were then centrifuged twice at 2004. The wild-type;21U sensor and pid-1(xf14);21U sensor libraries (oxidized and nonoxidized) were prepared as described before , pid-1(xf35), and pid-1(xf36) libraries (TAP and/or CIP-TAP) were prepared as described in the Supplemental Material. Sequencing data are available at Gene Expression Omnibus, entry GSE55309.Small RNA was isolated with a Mirvana kit from Life Technologies. Following isolation, this RNA was treated with TAP and calf intestinal phosphatase followed by TAP (CIP-TAP) or NaIOd before . Wild-ty


MSY2 and JHDM2A genes have animportant role in spermatogenesis process and fertility of male mice. But the potentialrole of these genes in human spermatogenesis and fertility is not known yet. Therefore,we evaluated expression ratios of these genes in testis tissues of men with normal andimpaired spermatogenesis. Animal model studies have shown that  In this experimental study, after RNA extraction and cDNA syn-thesis from 50 non-obstructive azoospermic and 12 normal testis tissues, the expressionratios of genes were evaluated by real time polymerase chain reaction (PCR) technique.Hematoxcylin and eosin (H&E) staining was used for histological classification of testis tissues. For statistical analysis, one way analysis of variance (ANOVA) test was carried out.YBX2 in sampleswith impaired spermatogenesis (p<0.001) compared to samples with qualitatively normal spermatogenesis and normal spermatogenesis; however, in JHDM2A gene, despitesensible reduction in gene expression level in men with impaired spermatogenesis, nosignificant differences were shown (p>0.05). Furthermore in YBX2, a significant negativecorrelation was demonstrated between the efficiency score of spermatogenesis and thethreshold cycle (CT) , whereas in JHDM2A, this negative correlationwas not significant . Our results showed a significant reduction in mRNA level of YBX2 and JHDM2A genes may playan important role in male infertility, and suggested that these molecules can act asuseful biomarkers for predicting male infertility.Generally, these data indicated that  YBX2 and JHDM2A genes are germ-cell-specific molecules which are essential for the production of functional spermatozoa; therefore, their inactivation could lead to male infertility. Y box binding protein 2 ( YBX2 ), also known as Contrin, is a human homologue of Xenopus DNA/RNA-binding and mouse MSY2 proteins , also known as JMJD1A or KDM3A, has been identified as an H3K9 demethylase ( for monomethylation and dimethylation ), while it has been originally cloned as a testis-specific gene transcript  and protamine proteins, while this gene is necessary for the proper chromatin reorganization during spermatid maturation by directly promoting the transcription of the transition nuclear protein 1 ( TNP1 ) and protamine1 ( PRM 1 ) genes  genes ,9. SinceThis experimental study was approved by the Ethical Committee of the Faculty of Medical Sciences of Qazvin University of Medical Sciences, Qazvin, Iran. Sampling was done under supervision of urologist in Fertility and Infertility Center of Shariati Hospital., Tehran, Iran, during 2012. After patients gave an informed written consent, testicular biopsies were obtained from 50 azoospermic men with mean age of 31.3 ± 3.7 years old; these patients were candidates of assisted reproductive technique (ART) and exhibited impaired spermatogenesis. In 12 patients with obstructive azoospermia after vasectomy, biopsies were carried out for diagnostic reasons during vasectomy reversal. These biopsies revealing normal spermatogenesis were served as controls, while the mean age of these individuals was 35 ± 2.9 years old. The limitation of this study was scarcity of tissue samples with normal spermatogenesis, for this reason sampling time was prolonged.To decrease possible confounding factors, the patients were excluded for these criteria: Y chromosome microdeletion, cystic fibrosis, varicocele, Klinefelter syndrome, or exposure to chemotherapy and radiation. In infertile patients with non-obstructive azoospermia, one part of testicular tissue specimens was used for testicular sperm extraction, while the other part was cut into two pieces; one piece was immediately prepared and frozen for RNA extraction procedure, and the other piece was fixed in Bouin’s fixative  and embedded in paraffin.For histological evaluation, 5-μm-thick paraffin sections were stained in hematoxylin and eosin (H&E), and then scored according to the modified Johnsen scoring system. In this system of classification, all tubular sections in each piece of the testicular biopsy are evaluated systematically, and each is given a score from 1 to 10 as follows: score 10 including complete spermatogenesis with many spermatozoa; score 9 including slightly impaired spermatogenesis, many late spermatids, and disorganized epithelium; score 8 including less than five spermatozoa per tubule and few late spermatids; score 7 including no spermatozoa, no late spermatids and many early spermatids; score 6 including no spermatozoa, no late spermatids and few early spermatids; score 5 including no spermatozoa or spermatids and many spermatocytes; score 4 including no spermatozoa or spermatids and few spermatocytes; score 3 including only spermatogonia; score 2 including no germinal cells andSertoli cells only; as well as score 1 no seminiferous epithelium .In summary, frozen testis tissues are homogenized in lysis buffer  using an ultrasonic processor UP100H  , and RNA is then extracted using RNeasy Mini Kit . In this regard, 1 volume of 70% ethanol was added to the homogenized lysate, and up to 700 μl of the sample was then transferred to an RNeasy spin column placed in a 2 ml collection tube. We used column DNase digestion to remove the residual DNA. After using different washing buffers, the RNeasy spin column was placed in a new 1.5 ml collection tube, 30-50 μl RNase-free water was added directly to the spin column membrane to elute the RNA, and finally RNA was frozen at -80˚C. Quality and quantity of isolated total RNA was measured using Nano Drop 2000c . In this case, RNA samples with A260/A280 ratios of >2 were selected for quantitative analysis. First strand complementary DNA (cDNA) synthesis was also performed using the RevertAid First Strand cDNA Synthesis Kit . For cDNA synthesis, 1-5 μg total RNA and 0.5 μg oligo (dt) were added to sterile, nuclease-free tube on ice, and total volume reached to 11.5 μl with DEPC-treated water. This solution was mixed and centrifuged briefly and then was incubated at 65˚C for 5 minutes. Next 4 μl 5X reaction buffer for reverse transcription, 0.5 μl RiboLock™ RNase inhibitor, 2 μl dNTP mix and 1 μl revertaid™ H minus reverse transcriptase  were added to solution, and total volume reached to 25 μl with DEPC-treated water. After being centrifuged, the solution was incubated for 60 minutes at 42˚C. Ultimately the reaction was terminated by heating at 70˚C for 10 minutes.YBX2 and JHDM2A) and GAPDH  were amplified with appropriate primers and probes   .Statistical analysis including mean, standard deviation (SD), and correlation coefficients (R) were done using Prism (version 3) software .Additionally, one-way analysis of variance (ANOVA) was carried out to determine the significant differences between the studied groups. A p value of <0.05 was considered as statistically significant.YBX2 (2-ΔCt: 0.5 ± 0.7) when compared to the samples with normal spermatogenesis (group I) (2-ΔCt: 3.6 ± 1.8) and qualitatively normal spermatogenesis (group II) (2-ΔCt: 3.03 ± 1.5). In the testicular tissues of azoospermic men with impaired spermatogenesis, YBX2 transcripts were 7.2 folds less than testicular tissues of men with normal spermatogenesis (-ΔCt) of YBX2 between the samples of group I and group II (p>0.05). The mRNA content of JHDM2A in testis tissues of studied groups showed that despite sensible reduction in gene expression in samples with impaired spermatogenesis (2-ΔCt: 0.17 ± 0.1), compared to samples with normal and qualitatively normal spermatogenesis , there was no significant difference in 2-ΔCt ratio among the three studied groups , whereas in JHDM2A, this negative correlation was not significant  exhibited a significant (p<0.001) lower mRNA level of ogenesis , but the p>0.05, . A signiYBX2 was seen significantly in comparison to normal ones in groups with different levels of impaired spermatogenesis . The proteins encoded by these two genes express specifically and highly in testicular tissue and are involved in functional regulation of some strategicmeiotic and post meiotic genes. Contrin ( YBX2 ) is a transcription and translation regulatory factor and a germ-cell-specific molecule which is essential for the production of functional spermatozoa. Generally 5% of adult human males are affected by male infertility, whereas, 75% are diagnosed as idiopathic due to unknown molecular mechanisms underlying the defects . Spermatin vitro assay studies showed that MSY2 acts as a transcription factor and an mRNA stabilizer which regulates expression of some testis specific genes in transcription and translation level. In this regard, MSY2 marks specific mRNAs ( those transcribed from Y-box promoters ) in the nucleus for cytoplasmic storage, thereby links mRNA transcription and storage/translational delay. In this process, MSY2 recognizes the CTG ATTGGC/ TC/TAA sequence as a DNA motif in the promoter of many genes that are specifically expressed in male germ cells. After MSY2 binds to its consensus promoter sequence, binds to transcripts of this gene. Subsequently this protein stabilizes and represses their translation as RNAprotein complexes (About the molecular function of this protein, animal models and omplexes ,17. MSY2-null mice. They found that the mutant males had an abnormally high number of apoptotic meiotic spermatocytes, lacked spermatozoa in the epididymis, and were sterile. Their results emphasized on the major role of this protein in male fertility. Meng et al. (YBX2 gene alterations in blood samples of men with severe defects in spermatogenesis, including azoospermia, severe oligozoospermia, and abnormal protamine expression. Their results showed seven polymorphisms were present at a statistically higher frequency in patients with infertility, particularly in men with abnormal protamine expression. This study showed t hat YBX2 gene has a potential role in male spermatogenesis, leading to male factor infertility (YBX2 in our samples, indicating the role of this gene in human spermatogenesis. Other regulatory factor which was studied in this research was JHDM2A gene. Okada et al. (JHDM2A gene with RNA polymerase II indicates that this gene may contribute to transcriptional activation of some testis specific genes. JHDM2A also stimulates TNP1 and PRM 1 genes, by bonding to core promoter and removing H3K9 methylation. Histone demethylase JHDM2A is critical for TNP1 and PRM 1 transcription and spermatogenesis (Jhdm2a-deficient mice have shown infertility and smaller testes (JHDM2A in studied samples, this correlation was not significant. Probably the role of this gene is not very influential in human fertility; therefore, more samples must be studied. Yang et al.  generateg et al.  and Dengg et al.  reportedg et al.  investigertility . Our resertility ,Table 2.a et al.  originalogenesis . Jhdm2a-r testes . DespiteYBX2 gene in testis tissue of azoospermic men, but future studies are needed to investigate the role of YBX2 and JHDM2A genes in regulating some specific genes of testis involving in human meiotic and post meiotic. Our findings indicated significant down-regulation of


Accretion rates varied substantially within and between islands, reef zones, levels of wave exposure, and island geomorphology. For forereef sites, mean accretion rates were the highest at Rose Atoll, Jarvis, and Swains Islands, and the lowest at Johnston Atoll and Tutuila. A comparison between reef zones showed higher accretion rates on forereefs compared to lagoon sites; mean accretion rates were also higher on windward than leeward sites but only for a subset of islands. High levels of spatial variability in net carbonate accretion rates reported herein draw attention to the heterogeneity of the community assemblages. Percent cover of key early successional taxa on CAU plates did not reflect that of the mature communities present on surrounding benthos, possibly due to the short deployment period (2 years) of the experimental units. Yet, net CaCO3 accretion rates were positively correlated with crustose coralline algae (CCA) percent cover on the surrounding benthos and on the CAU plates, which on average represented >70% of the accreted material. For foreeefs and lagoon sites combined CaCO3 accretion rates were statistically correlated with total alkalinity and Chlorophyll-a; a GAM analysis indicated that SiOH and Halimeda were the best predictor variables of accretion rates on lagoon sites, and total alkalinity and Chlorophyll-a for forereef sites, demonstrating the utility of CAUs as a tool to monitor changes in reef accretion rates as they relate to ocean acidification. This study underscores the pivotal role CCA play as a key benthic component and supporting actively calcifying reefs; high Mg-calcite exoskeletons makes CCA extremely susceptible changes in ocean water pH, emphasizing the far-reaching threat that ocean acidification poses to the ecological function and persistence of coral reefs worldwide.This paper presents a comprehensive quantitative baseline assessment of  As atmospheric CO2 dissolves in seawater, it forms the weak carbonic acid (H2CO3), which in turn dissociates into bicarbonate (HCO3−) and carbonate (CO32−) ions, and the associated protons (H+). Natural processes including gas exchange, photosynthesis, respiration, calcium carbonate (CaCO3) precipitation, and dissolution, influence the distribution of chemical species of the carbonate system as a function of pH ; analysediment; . Data fr3 cm-2 yr-1 at JOH-10, a lagoon site at Johnston Atoll, to 0.251 g CaCO3 cm-2 yr-1 at JAR-01 on the forereef at Jarvis Island. Of the 78 sites examined, average accretion rates differed between islands (n = 11) and reef zones (forereef vs. lagoon). There was no interaction, but each factor had a significant main effect, with rates being significantly greater at forereef sites compared to lagoon sites   did not differ from the equatorial reef systems above mentioned . In addition, urbanization and human inhabitation did not have a clear effect on the inter-island patterns of CaCO3 accretion. Although Johnston Atoll, Palmyra Atoll, and Tutuila exhibited the lowest island/atoll-wide accretion rates and coincidentally have undergone the greatest levels of human disturbance , the pattern of inhabitation/high disturbance regime and low accretion rates was not consistent for other inhabited islands such as Swains, Ta`u and Ofu-Olosega or historically human impacted reef systems such as those at Howland, Baker, and Jarvis. This is likely due to the low levels of human inhabitation at Swains, Ta`u and Ofu-Olosega    ; missing CAUs occurred haphazardly across a variety of sites including forereef, lagoon, sheltered, and exposed sites. Rose Atoll and Ta`u had the highest percentage of missing CAUs, with 10 and 15% of units missing, respectively. Net accretion rates varied across a wide range of spatial and environmental constructs including reef zone (forereef vs. lagoon), latitude, island, exposure (leeward vs. windward), population (urban settlements vs. none), geomorphology (carbonate vs. volcanic), and sites . Individon sites  (two-way p<0.01) . Differereau 2010. Conside= 0.027) . For the= 0.002) . At carb= 0.002) .2 = 13.31, df = 1, p<0.001) (3 sediment (4.4%), and calcified algal crusts (4.1%). This contrasts with the benthic communities at the deployment sites, where the major calcifying taxa included scleractinian corals (32%), CCA (26%), and calcified algae . For all reef systems with the exception of Johnston Atoll, CCA represented more than 50% of cover on the CAU plates and differences in CCA cover among islands were statistically significant    , as wellp<0.001) . Interesarisons) .in situ total alkalinity and salinity , and a negative statistical association with chlorophyll-a concentration ; mean Island accretion rates exhibited non-significant correlations all the other water chemistry parameters , as was CCA cover of the benthos  . Despiterameters .Halimeda  and non-coralline encrusting macroalgae ] and two were negatively correlated turf algae  and fleshy upright macroalgae ]. Of these, Halimeda was the only statistically significant variable retained in the GAM analysis.The optimal abiotic REVS model corroborated the results from the independent correlation tests above. As such, the spatial variability in the carbonate accretion rates on forereefs was best explained by two environmental predictor variables: total alkalinity and chlorophyll-a . In the subsequent GAM analysis, only total alkalinity was retained as the explanatory variable. For the lagoon sites, the optimal abiotic REVS model contained two environmental predictor variables that were positively associated with the carbonate accretion rates: silicon hydroxide  and dissolved inorganic carbon . In the subsequent GAM analysis, only dissolved silica was retained as a statistically significant predictor variable. The biotic variables to best predict the spatial variation in carbonate accretion rates on forereefs included CCA cover and coral cover  however, due to the low level of association only CCA was retained as statistically significant predictor in the GAM analysis. Finally, for the lagoon sites, the optimal biotic REVS model identified four explanatory variables; two were positively correlated with carbonate accretion rates [Halimeda) out-weighed those depositing high Mg-calcite.When net site-specific accretion rates were combined with the percent cover of the different functional groups of known mineralogy recruited to the CAUs and on the benthos, high Mg-calcite was found to be the dominant carbonate polymorph of the reef early successional stages. For oceanic reef systems such as Howland, Baker, Jarvis, Johnston, Swains, Rose, and Palmyra, the net accretion of organisms depositing high Mg-calcite represented over 70% on the CAU plates, compared to ~30% on the benthos . These d3 accretion in situ across a diverse range of reef systems in the central and south Pacific and demonstrates that: 1) net carbonate accretion rates of early reef successional stages varied considerably across a wide range of spatial and environmental constructs, including island, site, reef zone, latitude, exposure (leeward vs. windward), population (urban settlements vs. none), and geomorphology (volcanic vs. carbonate); 2) CCA benthic percent cover of the surrounding benthos, total alkalinity, and chlorophyll-a concentrations were significant predictor variables for net carbonate accretion rates on forereef habitats, and dissolved silica and percent cover of Halimeda were the significant predictor variable for lagoon habitats, respectively; and 3) the composition and relative abundance of the key early successional taxa recruited on to CAUs differed from that of the surrounding, mature benthos, with the former being overwhelmingly dominated by crustose coralline algae . The results of this study also provide insight into CaCO3 accretion rates on standardized surfaces across an anthropogenic gradient, from relatively undisturbed, quasi-pristine coral reefs to human impacted .The large range of accretion rates within and among islands are likely the result of the complex and spatially variable nature of the physical and biological processes driving the structure and function of reef communities. Overall, accretion rates were higher on forereef sites than in lagoon habitats because of the higher amount of CCA present on CAUs from these reef zones. Although the lagoon environments at Johnston, Rose, and Kingman Reef are very different from each other, the observed forereef vs. lagoon differences are likely driven by key coral reef community structural determinants, including depth, light availability, wave exposure, as well as, the disparate levels of water circulation and flushing, turbidity and sedimentation, and productivity that characterize each reef zone , 64, 65.3 cm-2 yr-1 and 0.060 g CaCO3 cm-2 yr-1, respectively) and Rose Atoll the highest ; accretion rates for the islands of Swains, Ta`u, and Ofu-Olosega were comparable to each other (0.09–0.100 g CaCO3 cm-2 yr-1). Interestingly, the three reef systems exhibiting the lowest average accretion rates  have also, historically, experienced the highest levels of human impact. For example, Johnston and Palmyra atolls were extensively dredged and substantially modified to accommodate the operation of military naval bases and air strips during the WWII U.S. Pacific campaign. Some of these alterations resulted in widespread, chronic changes to water clarity and circulation, in addition to more recent human disturbances including localized iron-leaching from ship groundings and PCB contamination [With the exception of Palmyra Atoll, the equatorial reef systems at Howland, Baker, Jarvis, and Kingman, exhibited comparable net carbonate accretion rates. In contrast, carbonate accretion rates in American Samoa and Johnston Atoll exhibited notably high levels of variability. Johnston and Tutuila at 16°N and 14°S, respectively, had the lowest average accretion rates  in seawater; benthic and water-column processes, including biological calcification and photosynthesis can drive site-level changes in carbonate ion concentrations [3 precipitation. In addition, Chl-a concentration was the optimal biotic predictor variable of net accretion rates at forereef sites. As previously mentioned, high nutrient concentrations, in particularly phosphate, have a detrimental effect of CCA calcification and growth [3 accretion rates of early reef successional stages.The spatial variability in CAU net accretion rates at forereef sites was related to total alkalinity (TA). TA, defined as the stoichiometric sum of the bases in solution, is a measure of the capacity of water to resist changes in pH. In tropical reef ecosystems TA is predominantly governed by the concentration of the carbonate ion , of which only dissolved silica was retained in the GAM analysis as a significant predictor variable. This finding is consistent with the selection of percent  systems , it was The average percent cover of the main benthic components at the study sites was approximately: 33% for scleractinian corals, 26% for CCA, and 16% for turf algae; on the CAU plates these taxa represented 0.4%, 70%, and 7%, respectively. It is the disparate proportions in percent cover of the key early successional taxa on CAU plates and the mature benthos the main reason why the RELATE analysis showed little similarity between CAU plates and the surrounding benthos. This can be explained in part due to the short deployment period (2 years) of the experimental units. Nonetheless, despite those differences, the spatial variability in carbonate accretion rates reported in this study could be predicted by the combination of biotic and abiotic parameters, demonstrating the utility of CAUs as a monitoring tool for the effects of ocean acidification (OA).Halimeda) out-weighed those depositing high Mg-calcite. Many coralline algal species precipitate high Mg-calcite [3 ratio at low latitudes and warm temperatures [3, making coralline algae amongst the most susceptible coral reef taxa to ocean acidification [In addition, high Mg-calcite was found to be the dominant carbonate polymorph deposited on the CAU plates. This is expected, given that CCA was the major calcifying functional group recruiting to the CAU plates, in contrast to the reef benthos where organisms depositing aragonite  under acidified conditions CCA may lose their competitive advantage as the dominant calcifying taxa of the early reef successional community, which in turn may have adverse implications for the settlement and development of other important reef calcifying taxa. Therefore, under the projected changes in marine seawater carbonate chemistry, the ability of marine biomineralizers to cope with such changes and continue offering the ecosystem services they currently provide will likely be determined by both the magnitude and rate of seawater pH decrease. As such, the combined effects of chronic human disturbances together with decreased pH from ocean acidification will likely affect reef community structure and therefore carbonate accretion on coral reefs worldwideOur study provides insight into variation in carbonate accretion rates, primarily by CCA, at dozens of sites across the central and south Pacific, and offers a unique perspective to contextualize our comprehension of the effects of OA at different scenarios of future ocean chemistry. As such, three main inferences can be gleaned from our observations: (1) the spatially variable nature of the accretion rates reported herein suggest that reef community responses will likely vary widely between reef systems, but between sites within islands as well; (2) because CCA precipitate a highly soluble polymorph of CaCO


Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication. Many plus-strand RNA viruses encode a viral RNA polymerase and capping enzymes to synthesize a 5’-capped RNA genome. However, how these two activities are coordinated during viral replication is not understood. In flaviviruses, polymerase and capping enzymes are encoded in a single multifunctional protein, where separate domains within the polypeptide are responsible for these activities; flavivirus NS5, composed of the polymerase and methyltransferase domains, carries out viral RNA synthesis, 5’-RNA capping, and RNA cap methylations. Previous NS5 monomer structures were unable to provide mechanistic insight into how the two domains communicate or the quaternary organization of the functional enzyme. We have determined the crystal structure of dengue virus NS5 and show that the NS5 dimer is likely the biological assembly of NS5, and RNA synthesis and RNA capping may be coordinated by the dimer. We found that essential interactions between the two NS5 domains can be maintained either within a monomer or via inter-molecular interactions within a dimer, and thus NS5 can adopt multiple conformations while preserving necessary interactions between the methyltransferase and polymerase domains. Using dengue virus, we additionally determined that such specific interaction between the two NS5 domains is the major determinant of viral replication. Flaviviridae includes at least 70 mosquito- and tick-borne viral species, some of which act as the etiological agents of human diseases such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV). Dengue is the most prevalent mosquito-borne virus, with nearly 400 million annual cases worldwide . NS5 was purified first by affinity chromatography using TALON cobalt affinity resin (Clontech) and an imidazole gradient of 5 to 150 mM in elution buffer . Fractions containing NS5 were concentrated to ~1 mL and further purified by size exclusion chromatography using a HiLoad 16/60 Superdex 200 preparative grade column  in 20 mM Tris-HCl pH 7.0, 300 mM NaCl, and 1 mM DTT. NS5 containing fractions were pooled. The protein concentration was determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific) with a theoretical molar extinction coefficient of 217,000 M-1cm-1 and a molecular weight of 104,000 Da.DENV3 NS5 constructs Δ2 and Δ6 with the priming loop shortened by either two , 500 μM (each) ATP, CTP, and UTP, 10 μM unlabeled GTP, 10 μCi of [α-32P]GTP, and 100 μM DTT. The subgenomic RNA template (719 nt) contains both 5’- and 3’-UTR regions of the DENV2 genome. The reaction was started by adding 10 μg (100 pmol) of purified NS5 before incubation at 37°C for 1 h. The reaction was terminated by acid phenol-chloroform extraction, followed by purification on a Bio-Rad P-30 column to remove the unincorporated nucleotides. Radioactive RNA products were analyzed by formaldehyde-agarose gel electrophoresis and visualized by autoradiography. Band intensities were measured with a PhosphoImager (Molecular Dynamics).Polymerase activity assays for wild-type, Δ2, and Δ6 NS5 were performed using the subgenomic RNA template as previously described . BrieflyNS5-Δ2 and Δ6 proteins were concentrated to ~10 mg/mL and screened using the sitting drop vapor diffusion method in 96-well plates using a Phoenix RE liquid handling robot (Rigaku). Several conditions produced small crystals within 2 weeks for Δ2 and Δ6, which were further optimized. The best-diffracting NS5-Δ6 crystals were lens-shaped, and grew to full size within 3–7 weeks in 1.0 M succinic acid (pH 7.0), 0.1 M HEPES pH 7.0, and 1% PEG monomethylether 2000. Crystals were harvested by cryo-cooling in liquid nitrogen after soaking for ~20 seconds in well solution supplemented with 25% ethylene glycol.1/2 and CC* values of 0.157 and 0.520, respectively, in the highest resolution shell (3.66–3.60 Å) [P3221 with a = b = 215.3 Å, c = 480.7 Å, and contained eight NS5 monomer in the ASU with a corresponding solvent content of 68%. The initial structure solutions were obtained by molecular replacement with DENV3 MTase  and RdRp  as search models using the program PHASER in the Phenix suite [2Fo-Fc map were clearly visible between the C-terminus of MTase and the N-terminus of RdRp, indicating which copies of the MTase and RdRp were part of each of eight intact polypeptide chains. The distance between the terminal ends of each corresponding MTase/RdRp pair was ~24 Å (distance between Cα atoms of R262 and N272), excluding the possibility of any other domain arrangement and making the assignment of continuous protein chains unambiguous. The 2Fo-Fc map also indicated that either SAM or SAH is bound to the MTase active site. We modeled SAH in the density, because SAH was copurified in several high-resolution MTase structures [X-ray diffraction data were collected at 100 K at the Advanced Photon Source beamline 21 . Four datasets were collected from three crystals. Reflections were indexed and integrated using HKL2000, and four datasets were scaled and merged together using SCALEPACK . A 3.6 Å–3.60 Å) . The cryix suite . Seven Mructures ,50, and ructures , zinc atfree factors of 23.8 and 27.4%, respectively  in the presence of the 7-MeGpppG cap analog.Construction of a full-length cDNA of DENV2 (New Guinea C strain) and its NS5 chimera containing the DENV4 MTase in yeast/escribed ,55. The 6 cells were resuspended in 100 μl of Ingenio solution . After pulsing, cells were carefully transferred into prewarmed complete medium (Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum and 1× streptomycin/penicillin), and allowed to recover for 5 min at 37°C in an incubator. Cells were resuspended in 10 ml of complete DMEM and incubated in a T-12.5 flask. On days 2 and 9, cells were trypsinized and transferred into a T-25 and T-75 flask, respectively. This procedure was repeated using one-third of the trypsinized cells from a T-75 flask every 5–7 days. For immunofluorescence assay, RNA-transfected cells at the end of indicated time periods were seeded into a slide (LabTek), and fixed by treatment with acetone. Cells were incubated with a 1:200 dilution of 7E11, a monoclonal antibody against DENV2 NS1. Fluorescein isothiocyante (FITC)-labeled, goat anti-mouse immunoglobulin G conjugate (Kirkegaard & Perry Laboratories) was used as a secondary antibody at a 1:100 dilution. Immunofluorescence photomicrographs (×200 magnification) were acquired using a Leitz Diaplan microscope coupled to the Leica/Wild MPS48 automated photographic system. The numbers and intensities of positive cells were compared utilizing the ImageJ program , as previously described.The DENV2 RNA (∼3 μg) containing either the wild type (DENV2) NS5, a NS5 chimera (DENV4 MTase and DENV2 RdRp), or DENV4 NS5 were transfected by electroporation  into BHK-21 cells , as previously described . Brieflyhttp://www.ncbi.nlm.nih.gov/genome/viruses/variation/dengue/) was used to remove residues that have some variations in each serotype [Serotype-specific residues were identified in two steps. First, seven to ten NS5 sequences from each DENV serotype were randomly selected for multiple sequence alignment using Clustal W . DENV1 Nserotype .S1 FigEach of the four dimers within the ASU and a symmetry-generated dimer from PDB entry 4V0Q are shown in gray as a molecular surface. Crystal contacts within 4 Å of molecules from neighboring ASUs in the crystal are indicated by a colored surface.(TIFF)Click here for additional data file.S2 FigFo-Fc omit map for SAH is shown as blue mesh contoured at 3σ. SAH and surrounding MTase residues are shown as yellow and cyan sticks, respectively. The RdRp domain and the linker are colored as in The (TIFF)Click here for additional data file.S3 FigInterface residues from MTase and RdRp fingers subdomain are shown as sticks and colored as in (TIF)Click here for additional data file.


Membrane transporters catalyze the transport of small solute molecules across biological barriers such as lipid bilayer membranes. Experimental identification of the transported substrates is very tedious. Once a particular transport mechanism has been identified in one organism, it is thus highly desirable to transfer this information to related transporter sequences in different organisms based on bioinformatics evidence.Escherichia coli, Saccharomyces cerevisiae, and Arabidopsis thaliana belong to the same families of the Transporter Classification (TC) system, and at what level these membrane transporters mediate the transport of the same substrate. We found that two membrane transporter sequences from different organisms that are aligned with normalized BLAST expectation value better than E-value 1e-8 are highly likely to belong to the same TC family (F-measure around 90%). Enriched sequence motifs identified by MEME at thresholds below 1e-12 support accurate classification into TC families for about two thirds of the sequences (F-measure 80% and higher). For the comparison of transported substrates, we focused on the four largest substrate classes of amino acids, sugars, metal ions, and phosphate. At similar identity thresholds, the nature of the transported substrates was more divergent (F-measure 40 - 75% at the same thresholds) than the TC family membership.We present a thorough benchmark at which level of sequence identity membrane transporters from We suggest an acceptable threshold of 1e-8 for BLAST and HMMER where at least three quarters of the sequences are classified according to the TC system with a reasonably high accuracy. Researchers who wish to apply these thresholds in their studies should multiply these thresholds by the size of the database they search against. Our findings should be useful to those who wish to transfer transporter functional annotations across species. Prokaryotic and eukaryotic genomes each encode for hundreds of membrane transporter proteins that play essential roles for the cellular import and export of ions and small molecules. Furthermore, transporters mediate signal transduction processes catalyzing the export and uptake of signaling molecules. Therefore, the functional classification of membrane transporters is an important task. The available experimental knowledge about transporter function has been compiled in databases such as TCDB , TranspoThe TC system categorizes transporter sequences according to their class, subclass, (super) family, and subfamily on the basis of functional or phylogenetic information that is based on sequence similarity. An example for this classification would be the PTS Glucose-Glucoside (Glc) super family 4.A.1 that belongs to class ‘4’ group translocators and subclass ‘A’ phosphate transfer-driven group translocators. Subfamilies might correspond to transported substrates. A particular transporter sequence in such a family is identified by an extra digit to the right as e.g. 4.A.1.1.1.Arabidopsis thaliana. In fact, many databases ignore the fourth digit (subfamily) of the TC system that normally refers to the main substrate. Schaadt and Helms have recently reported that membrane transporters from Arabidopsis thaliana that either transport amino acids, oligopeptides, phosphate, or sugar molecules can be distinguished from each other based on their amino acid composition Ec (155),nsportDB , respectdatabase  for the Ec), 29 (At), and 34 (Sc) different TC families. Subclass 2.A  and subclass 3.A (P-P-bond-hydrolysis-driven transporters) were the most common TC subclasses. In Sc and At, the Major Facilitator Superfamily 2.A.1 accounts for nearly 40% of all transporters while in Ec it is the second largest family after the ATP-binding Cassette (ABC) Superfamily 3.A.1. Shared TC families belong mostly to TC classes Electrochemical Potential Driven Transporters (class 2) and the Primary Active Transporters (class 3).The transporters of the three organisms are annotated to 53 (Hs). Sugar, amino acid, and metal transporter sets were extracted from the ChEMBL database [For the testing part, we created four datasets of experimentally annotated human transporters , 14 for (Sc- Ec), and 12 for (At- Sc). BLAST E-values were normalized by the number of residues in the searched database (see Results section). HMMER E-values were normalized by the number of hits.First, we used NCBI BLAST version 2.2.23 and HMMER version 3.0 for pairwise comparisons of all 90 In order to identify reliability thresholds at which functional information can be safely transferred between organisms, we tested thresholds  for the E-values and evaluated prediction accordingly. We calculated the accuracy measures precision , recall (sensitivity) and F-measure  at each threshold to evaluate the prediction performance Tables , 6 and 7Ec: Escherichia coli; Sc: Saccharomyces cerevisiae; At: Arabidopsis thaliana.The authors declare that they have no competing interests.AB and VH conceived this study. AB wrote scripts, compiled data-sets, and performed the data analysis. AB and VH analyzed the data and jointly wrote the manuscript. Both authors read and approved the final manuscript.Considered transporters in organisms EC, AT and SC respectively.Click here for fileHs, At) and .Complete annotation results of the pairs (Click here for fileResults of FASTA global searches.Click here for fileHeatmap of BLASTing Ec substrate-TC families against At families.Click here for fileHeatmap of BLASTing Ec substrate-TC families against Sc families.Click here for file


NL4-3, SIVcpz, and SIVmac . Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1 Human immunodeficiency virus (HIV) is a persistent and critical threat to human health. Replication and pathogenesis of HIV is governed by information encoded in its single-stranded RNA genome. In addition to coding for viral proteins, the HIV genomic RNA forms base paired and higher-order structures that are critical for viral replication. It is likely that only a subset of functional RNA motifs has been identified. Here, we interrogate the structures of three diverse HIV-related viral genomes by nucleotide-resolution chemical probing. The three genomes include HIV-1, the virus that infects humans, and SIVcpz and SIVmac, which are progenitors for the main branches of the two HIV evolutionary groups. We used a structure-informed alignment approach to generate consensus models for base-paired secondary structures that are shared by these three HIV-related genomes. With this approach, we were able to recapitulate all known RNA structures and, additionally, discovered multiple previously undescribed structural elements that are clearly conserved among major HIV groups. We anticipate that the methods described here will be broadly useful for RNA structure motif discovery and, more immediately, for identification of RNA targets in HIV that are promising sites for therapeutic intervention. RNA plays a direct role in most biological processes , and mulenv gene, the Rev response element (RRE) binds the viral Rev protein, allowing unspliced and partially spliced viral mRNA to be exported out of the nucleus , and the resulting solution was characterized by absorption spectroscopy at 260 nm to determine RNA concentration.2 were added, and the RNA solution was brought up to a volume of 90 μL using genome resuspension buffer. The RNA solution was then incubated at 37°C for 15 min before adding 10 μL of 100 mM 1M7 in DMSO (1M7 modification of natively folded RNA) or 10 μL neat DMSO (background control). The RNA solution was then incubated at 37°C for 3 min to allow complete reaction of 1M7. The RNA solution was then held on ice until purification.To determine SHAPE reactivities, three experiments were performed with SIVcpz and SIVmac samples: 1M7 modification of natively-folded RNA, a no-modification background control, and 1M7 modification of denatured RNA . For 1M7For 1M7 modification of denatured RNA, an aliquot containing 1 μg of SIVcpz or SIVmac RNA was taken from precipitated RNA stocks. To this aliquot, 25 μL of 4 denatured control buffer was added , and the RNA solution was brought to a volume of 40 μL using nuclease-free water. To this RNA solution, 50 μL of deionized formamide was added. The RNA solution was held at 95°C for 1 min and then added to 10 μL 100 mM 1M7 in DMSO. The reaction was held at 95°C for 1 min before transferring the reaction to ice. The RNA solution was held on ice until purification.RNA was then purified by affinity binding . Following purification, sequencing libraries were generated as described , and seqSHAPE-structure dependent alignments were performed as described . PairwisY represents HIV-1 SHAPE values and X1 and X2 represent SIVcpz and SIVmac SHAPE values, respectively:SHAPE data were fit using least squares, where Correlations between the three HIV-related strains were evaluated using the F-test, which evaluates the interdependency of data sets based upon a linear regression model. The F-test evaluates the following null hypothesis by the sum of squares due to lack-of-fit for the model:p-values defining the significance of the interdependence of SHAPE values were determined over the entire alignment. In addition, t-tests were used to evaluate pairwise correlations between HIV-1 and either SIVcpz or SIVmac. Statistical analyses were performed over 200-nt windows in the multiple sequence alignment. Over each window, only positions with SHAPE values for each genome were considered; no gapped positions were included. Multi-variable linear regression analyses were performed using the NumPy, SciPy, and statsmodels Python modules [Based on the derived F-statistic measurements,  modules , 45. MulA manually edited alignment considering diverse primate lentiviruses was taken from the 2012 HIV Compendium (section “Alignment of Primate Lentivirus Complete Genomes”) . A referAreas of interest for secondary structure modeling were selected based on F-test statistics of multi-variable linear regression models. If a given 200-nt window had an F-test p-value less than 0.01, the corresponding 200-nt region was selected as an area of interest. Consecutive areas of interest were combined in the same secondary structure element.Secondary structures for the reference HIV-1 sequence were generated with a two-step procedure using RNAalifold and RNAfold, both of the Vienna-RNA software package , 32, 48.i) pairs with shared nucleotides contradicted each other in terms of base pairing partners or (ii) pairs from two consensuses were non-nested. HIV-1 structure predictions constrained by consensus pairs were performed with RNAfold. Predictions were constrained such that curated consensus pairs were maintained in the final structure. SHAPE reactivities were incorporated into secondary structure model using a pseudo-free energy potential [Following consensus model generation, consensus base pairs were used to constrain a single-genome secondary structure prediction for HIV-1. Base pairs from each consensus structure with pairing probabilities greater than 95% were added to a constraint list. The constraint list was curated such that consensus pairs were excluded if either  recapitulated in a given alignment, for the SHAPE-dependent alignment relative to the LANL alignment is shown across the entire HIV-1 genome over 200-nt windows. HIV-1/SIVcpz and HIV-2/SIVmac reading frames and the positions of (EPS)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.