Increasing evidence demonstrates that hepatitis E virus (HEV) can be transmitted across species. According to previous reports, swine HEV has two genotypes, genotype 3 and 4, and both can infect humans by the fecal-oral route. Thus, it is crucial for the control of HEV zoonotic transmission to evaluate the dynamics of viral shedding and distribution in different tissues during cross-species infection by HEV. In this study, rabbits were infected with genotype 4 swine HEV by the intraperitoneal route. The results showed that HEV RNA not only shed in the feces but also in the saliva of some rabbits during infection with swine HEV. Viremia appeared late after infection, and anti-HEV IgG was not obvious until the appearance of high viremia levels. After the rabbits were euthanized, a histopathological examination showed that the livers developed overt hepatitis accompanied by an elevation of alanine aminotransferase (ALT) and aspartate transaminase (AST). Furthermore, HEV RNA was detected in various tissues, especially in the salivary glands and tonsils. Subsequently, negative-stranded HEV RNA was practiced in tissues with positive HEV RNA, which demonstrated that HEV replicated in the tissues. Next, we harvested additional tissues from the liver, salivary gland, tonsil, spleen, thymus gland, lymph node and intestine, which are known as replication sites of swine HEV. Additionally, we also observed the HEV antigen distributed in the organs above through immunohistochemical staining. These results demonstrate that rabbits could be used as an animal model for researching cross-species infection of genotype 4 HEV. It is also noteworthy that HEV can shed in the saliva and presents the risk of droplet transmission. These new data provide valuable information for understanding cross-species infection by HEV. Hepatitis E (HE) is a fecal-oral transmission disease caused by HEV, which is a non-enveloped, positive-sense, single-stranded RNA virus . HepatitAnimal models have been the main tools for researching HEV due to the lack of an efficient cell culture system . AlthougGrowing evidence has indicated that hepatitis E is zoonotic. Previous studies showed that HEV can be isolated from rats, boars, rabbits, ferrets, camels and cows –24. AddiTherefore, we explored whether rabbits can be infected with genotype 4 swine HEV by the intraperitoneal route and evaluated viral shedding in the feces and saliva. We then detected the levels of antigen, anti-HEV IgG and hepatic enzymes. The rabbits were sacrificed at 28 and 49 days post-inoculation (dpi), and replication sites and the location of the ORF 2 antigen were detected. The goal was to better understand the mechanisms underlying HEV cross-species infection.The animal experiment was approved by the Animal Care and Use Committee of China Agricultural University (CAU) (permit number: 20151110–160). We followed the guidelines of the CAU Animal Care and Use Committee in handling the experimental animals during this study.7 genome equivalents per mL.Strain HB-L3 of swine HEV, belonging to genotype 4 and having 90.9% homology with a Beijing human strain (GenBank No. AJ272108), was isolated and stored by our lab  . To geneSixteen 80-day-old female New Zealand white rabbits weighing between 1800 and 2000 g were purchased from the Xing Long Experimental Animal Center, Beijing, China. Before inoculation, the blood and feces of all the rabbits were confirmed to be negative for HEV RNA with reverse transcription-nested PCR (RT-nPCR). The sera were confirmed negative for HEV antigen and HEV antibody using enzyme-linked immunosorbent assay (ELISA).Sixteen rabbits were randomly divided into two groups of eight rabbits per group. Each rabbit in the experimental group was inoculated intraperitoneally with 10 mL of viral suspension per day for 7 consecutive days. Each rabbit in the control group was injected with an equal volume of negative fecal suspension. Each rabbit was housed in a separate cage and fed twice a day with access to drinking water. The condition of all the rabbits was monitored everyday, and no rabbit died following viral inoculation. No clinical symptoms were observed in the HEV-infected rabbits. All the rabbits were sacrificed by air-injection at the end of the experiment.Throat swab samples and feces were collected and tested for HEV RNA at the indicated times. Blood was collected and tested for HEV antibodies and antigens weekly to avoid animal stress. Half of the rabbits were euthanized at 28 dpi and the other half at 49 dpi. Their blood, bile, hearts, lungs, livers, kidneys, spleens, lymph nodes, thymuses, tonsils, salivary glands, stomachs, small intestines , sacculus rotundus (SR), cecum and appendices were collected, stored at -80°C for RT-nPCR and fixed in 4% paraformaldehyde for paraffin sections. The collected serum was tested for alanine aminotransferase (ALT) and aspartate transaminase (AST) levels at the Animal Hospital of China Agriculture University.All the serum samples were collected and tested for HEV antigen and anti-HEV IgG using an HEV ELISA kit. All assays were performed according to the manufacturer’s instructions , 16.™ RNA Kit ; then, the RNA was reverse transcribed using the HiFi script cDNA Synthesize Kit  according to the manufacturer’s instructions. Nested PCR was carried out to amplify the partial fragments of ORF2 using outer primers P1, P2 and inner primers P3, P4. The PCR parameters were described in Yang’s article [Total RNA was extracted from the collected samples using an UltraPure article .2O was included as the negative control.Samples with positive HEV RNA were assayed for negative-sense HEV RNA , 28. TheAll fixed tissues were processed routinely in paraffin, and 4-μm sections were prepared. The sections were stained with hematoxylin-eosin (H&E) stain for routine histopathological examination. Monoclonal mouse anti-HEV ORF2 and ORF3 antibodies  werethe primary antibody for immunohistochemistry, as described by Mao et al. .The sampFecal and throat swab samples from all rabbits in the control group were negative for HEV RNA during the study.Fecal samples from all rabbits infected with swine HEV were positive for HEV RNA at different time points. At 7 dpi, HEV RNA was first detected in the feces of T4. The feces of all the rabbits were positive for HEV RNA over time. The shedding of HEV RNA was not consecutive in the feces during the early period of infection. Shedding of HEV RNA usually lasts 3~7 days and reappeared after an interval of 7~10 days in the infected rabbits. During the later period of infection, HEV RNA disappeared in the feces of T8 rabbit .The throat swab samples from 3 rabbits were positive for HEV RNA during the study. Throat swab samples of T4, T5 and T6 rabbits were positive for HEV RNA at 24 dpi, 35 dpi and 38 dpi, respectively. Shedding of HEV RNA lasted for 3~10 days and disappeared. In the T5 rabbit, throat swab samples were positive for HEV RNA, but the feces were negative for HEV RNA .P values.At 28 dpi, the ALT and AST levels in the experimental group were significantly higher than in the control group (p<0.05). Dramatic elevation of ALT and AST in the experimental group was observed at 49 dpi. The average ALT and AST reached 96.3 and 102.1 U/L, respectively, in the experimental group . SPSS 20Viremia was first detected at 35 dpi in T5 rabbits and was elevated until 49 dpi. At 49 dpi, viremia was also detected in T6 and T8 rabbits. Anti-HEV IgG was only detected in the T5 rabbit at 42 dpi and remained elevated until 49 dpi . Both HEAll the tissues from rabbits in the control group were negative for HEV RNA throughout the study. At 28 dpi, HEV RNA was detected in various tissues, including liver, bile, salivary gland, spleen, thymus gland, lymph nodes, small intestine, SR, cecum, appendices, and kidney. At 49 dpi, HEV RNA was also detected in the blood, liver, bile, salivary gland, spleen, lymph nodes, small intestine, SR, cecum, appendices, heart, lung, and pancreas .The discovery of negative-stranded HEV RNA and ORF 3 antigen in the tissues demonstrates that HEV replicates therein ,32,33,34Various tissues in the control group revealed no obvious lesions . Livers Using immunohistochemical staining, HEV antigen was detected in the liver, salivary gland, tonsil, spleen, thymus gland, lymph nodes, small intestine , SR, cecum, appendices, kidney, heart, lung and pancreas. Positive signals were detected in the cytoplasm of hepatocytes and biliary epithelial cells; the duct epithelium of the salivary gland and pancreas; the crypt epithelium and macrophages or dendritic cells of lymphoid follicles in the tonsil, spleen, lymph node, SR, appendix, and intestinal mucosal epithelium; the follicle-associated epithelium of the SR and appendix; the thymic epithelial cells in the medulla, myocardium, the epithelium of the renal tubules and the epithelium of the bronchioles and alveoli . No posiAccording to previous reports, rabbits have been infected with swine HEV or genotype 4 HEV and developed hepatitis by intravenous inoculation , 32, 33.Rabbits infected with swine HEV had obvious HEV shedding in the feces and via throat swabs at different time points. Unlike the duration of fecal viral shedding in pigs and Mongolian gerbils, rabbits infected with swine HEV had intermittent fecal viral shedding in the early stage of infection , 15. HowCompared with normal HEV RNA detection in feces, the detection of HEV RNA in throat swab samples is interesting. HEV RNA was once detected in nasal swab materials in pigs experimentally infected with swine HEV, and it was thought that the pig snouts may have been contaminated with feces . AlthougUnlike Mongolian gerbils infected with swine HEV , rabbitsHEV RNA was detected in various organs, which was consistent with previous reports of animals infected with HEV by experimental or natural infection , 33, 41.In our study, obvious lesions in the liver and kidney were observed, which is consistent with previous studies , 41, 42.Moreover, HEV ORF2 antigen was mainly distributed in the duct epithelium in the salivary gland, pancreas and mucosal epithelium of the intestine according to immunohistochemical staining. This showed that the epithelium associated with the digestive system might be susceptible to HEV infection. Additionally, HEV ORF2 antigen was observed in macrophages or dendritic cells in the spleen, lymph node, tonsil, gut-associated lymphoid tissue (GALT), SR and appendix . This waIn summary, we demonstrated that rabbits can be infected with genotype 4 swine HEV and develop overt hepatitis. This means that rabbits could be used as a model for infection with genotype 4 swine HEV. Furthermore, detection of HEV shedding in saliva means a risk of droplet transmission, and this may be a new route of HEV transmission which needs further study. These results provide valuable information for the future exploration of the pathogenesis of HEV cross-species infection.S1 FigA. Liver. The positive signal for HEV antigen was detected in hepatocytes and the epithelium of bile ducts. B. Salivary gland. The positive signal for HEV antigen was detected in the duct epithelium of salivary gland. C. Tonsil. The positive signal for HEV antigen was detected in the crypt epithelium and in macrophages in the tonsil. D. Spleen. The positive signal for HEV antigen was detected in macrophages or dendritic cells in the spleen. E. Thymus gland. The positive signal for HEV antigen was detected in thymic epithelial cells in the medulla of the thymus gland. G. Small intestine. The positive signal for HEV antigen was detected in the mucosal epithelium in the small intestine. H. SR. The positive signal for HEV antigen was detected in macrophages or dendritic cells of lymphoid follicles. I. Appendix. The positive signal for HEV antigen was detected in the follicle-associated epithelium. The positive signal is yellow. (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants. Arabidopsis thaliana contains a total of 87 genes encoding mostly ribosomal and photosynthetic subunits, 4 rRNA and 37 tRNA genes [The plastid is the result of an endosymbiotic event that occurred billion years ago and comprised the incorporation of a cyanobacterium into a eukaryotic, mitochondria-possessing cell. The main benefit of the cyanobacterium for the host cell was the supply of additional energy and photoautotrophic growth, granting an enormous selective advantage. The genome of the endosymbiont underwent dramatic transformations, whereby the majority of the cyanobacterial genes were transferred into the nucleus or lost . MeanwhiNA genes .plastid-encoded bacterial type RNA polymerase (PEP) and at least one phage-type nuclear-encoded RNA polymerase (NEP). The PEP is composed of four basic plastid-encoded core subunits (rpo genes) and requires additional nucleus-encoded sigma factors (six in Arabidopsis) for promoter recognition and transcription initiation as well as further factors, which associate to the core during chloroplast development. In contrast, NEP is a one-subunit-enzyme which seems to require additional accessory factors as well. It was assumed that photosynthetic genes are predominantly transcribed by the PEP, while genes for the translational machinery and for PEP subunits are targets of the NEP [rpo genes and genes for the translational machinery, transcripts preferentially generated by the NEP predominate [Similar to the operons of the cyanobacterial ancestor, the chloroplast genes are primarily organized in newly composed transcriptional units and often contain genes that do not belong to the same functional cluster. These units are transcribed as polycistronic pre-mRNAs by two types of RNA polymerases: the  the NEP . However the NEP , whose r the NEP . In mostdominate .psbD–psbC promoter switch in response to blue light in barley, gene-specific transcriptional regulation seems to be an exception rather than the rule [Even though there are examples for gene-specific transcriptional regulation, such as the the rule ,12. Unlithe rule . Most obthe rule . These nthe rule . The prothe rule ,16–18.Arabidopsis, 17 gene products assigned to exhibit ribonucleolytic activity or to contain domains characteristic for ribonucleases were predicted to be localized in the chloroplast [Arabidopsis chloroplasts, RNR1 (RNase R) and PNPase with 3′→5′ specificity, and RNase J with 5′→3′ activity. As in other organisms, plant ribonucleases are thought to be rather unspecific and certain substrate preferences might be determined by the RNA structure or chemical features and less by the RNA sequence [The conversion of polycistronic transcripts into oligocistronic or monocistronic isoforms, the post-transcriptional generation of 5′ and 3′ transcript termini as well as RNA decay in general is accomplished by plastid ribonucleases . In chlooroplast . Among toroplast ,17, whiloroplast . CSP41 that cleave the RNA in unstructured, unprotected regions . AlthougMost of the plastid RNA protectors present in vascular plants are not found in green algae, such as Chlamydomonas, and vice versa, reflecting the diverged genome–plastome interaction and the tight coevolution of the cellular genetic compartments in the respective lineages during endosymbiosis. Comparably dissimilar are post-transcriptional processes in plastids of land plants and green algae. For example, RNA editing requires a plethora of trans-acting factors and is widespread in vascular plants but entirely absent in green algae . Often, pentatricopeptide repeat (PPR) proteins, (2) chloroplast ribonucleoproteins (cpRNPs), and 3) additional RNA protectors, which do not belong to a larger protein family. Our review article mainly focuses on vascular plant chloroplasts as gene expression in Chlamydomonas reinhardtii differs in many respects from vascular plants and extensive reports deserve an independent comprehensive review article. The reader is referred to recent articles reporting on plastid mRNA stability in this unicellular algae  ae e.g. ,33–41 ). .pentatrThe family of plant PPR proteins presumably evolved from tetratricopeptide repeat (TPR) proteins and consists of more than 450 up to approximately 1000 conserved members, shared among the eukaryotic kingdom ,43. PPR atpH and rpl33 as well as the 3′-UTR of atpI and psaJ, whose intergenic regions (atpI–aptH and psaJ–rpl33) share consensus sequences to which PPR10 specifically binds [psaJ, offering new RNA-binding regulation perspectives via different dimerization states [atpH by restructuring its 5′ region and exposing its otherwise masked ribosome-binding site [The best understood PPR protein is PPR10 in maize. PPR10 is essential for the stabilization of two different RNA termini: the 5′-UTR of ly binds   mutants were screened [hcf152 mutant showed defects in the processing and accumulation of the psbB–psbT–psbH–petB–petD transcript cluster accompanied by severe defects in PSII and cytochrome b6f complex formation [psbH–petB intergenic region in monocots and dicots [petB and psbH RNAs respectively, as revealed by precise transcript mapping [In vitro RNA-binding experiments validated that this sRNA represents the direct target of HCF152, suggesting that HCF152 protects the cleaved 5′ petB and 3′ psbH RNAs with identical sequences in their termini against exonucleolytic degradation, which resembles the function of PPR10 [In a first step to identify novel factors in screened . The hcformation ,56. Furtormation . A screed dicots ,50,58, s mapping . In vitrof PPR10 ,24.crp1 (chloroplast RNA processing 1) was identified in a screen for mutants with distinct defects in the expression of plastid-encoded genes. It was shown to lack the cytochrome b6f complex, which could be traced back to defects in the accumulation of monocistronic petB and petD transcripts [petA, petD, and psaC mRNAs [In vivo RIP-chip experiments with the 5′-untranslated regions of psaC and petA mRNAs further contributed to the understanding of CRP1 function as a translational regulator [petA 5′-UTR [Arabidopsis homolog AtCRP1 revealed an association with the 5′-UTR of psaC, the petB–petD intergenic region and to a much weaker extent the petA 5′-UTR [Arabidopsis and maize the overall analyses argue for similar functions of both CRP1 proteins in stabilization of petD 5′ and petB 3′ RNA termini and in translational regulation of psaC and petA mRNAs.The maize mutant nscripts . In addiaC mRNAs . In vivoegulator . ElectroA 5′-UTR . FurtherA 5′-UTR . This isA 5′-UTR . Taken tZea mays mutant ppr53 revealed that 5′ transcript ends of rrn23 and ndhA RNAs were absent. PPR53 was able to bind to the 5′ end of the rrn23 RNA, which was also predicted to be the PPR53 target site, but failed to do so in case of the 5′ end of ndhA RNA. Thus, the authors concluded that PPR53 stabilizes the 23S rRNA by binding to its 5′ region and stimulates the translation of ndhA via another yet unknown factor(s) [Arabidopsis ortholog SOT1 suggest that the binding of SOT1 to the 5′ end of the dicistronic 23S-4.5S rRNA prevents its exonucleolytic degradation [Analysis of the actor(s) . Experimradation . Moreoveradation .pgr3 alleles revealed defects in cytochrome b6f and NDH complex accumulation. These defects were traced back to impaired stabilization of the tricistronic petL–petG–psaJ RNA precursor and putative defects in the translation of petL and presumably a member of the ndh transcripts [in vitro studies revealed an association of recombinant PGR3 with the 5′-UTRs of petL and ndhA, arguing for a role of PGR3 in the stabilization and translational activation of these two RNA targets [petL and ndhA [Arabidopsis pgr3 mutants revealed that the defects in petL stabilization and translation are conserved. However, as pgr3 mutants in maize showed more dramatic effects than the Arabidopsis ones, it was concluded that ZmPGR3 has acquired additional functions compared with the Arabidopsis ortholog [Proton gradient regulation3 (PGR3) is an example for another PPR protein that harbors two functions: transcript stabilization and translation initiation. Initial experiments including different nscripts . Further targets . Finallyand ndhA . Compariortholog .trnG–UCC precursor [In vitro binding assays with recombinant ZmPPR5 could narrow down its binding site to the central region of the trnG–UCC intron, suggesting that PPR5 protects an endonuclease-sensitive site through its binding and might even facilitate the splicing of this group II intron [The stabilizing function of PPR proteins is not restricted to protein coding mRNAs and rRNAs but extends to tRNAs, as demonstrated for the maize PPR5 protein that associates with the recursor . In vitrI intron   mutant showed that rRNA processing and accumulation are affected causing a prevalent reduction of plastid proteins [atpA and atpB translation [atpF–atpA and psaJ–rpl33 intergenic regions, thus acting on similar RNA targets as PPR10. Differences in the mutant phenotype in monocot and dicot species argue for evolutionary diverged functions of ATP4 and SVR7 [Analysis of proteins . A laternslation . Additioand SVR7 .rps12–rps7–ndhB precursor RNA is likely to be processed at not less than two sites in the rps7–ndhB intercistronic region. RNase protection assays revealed that the PPR protein CRR2 is required for intergenic processing of the site close to ndhB [ndhB transcripts by binding to this region, thus hindering 5′-3′ exonucleolytic cleavage. This is reflected by the lack of 5′ processed ndhB transcripts whereas the precursor RNA accumulates at normal levels in crr2 mutants in Arabidopsis, which are unable to accumulate stable and functional NADH dehydrogenase-like complexes mediating cyclic electron flow around photosystem I [in vitro even if the domain was considered to be related to RNA editing [ndhB 5′-UTR has never been investigated, its potential binding site is presumably represented by an abundant RNA footprint downstream of the 5′ end of the monocistronic ndhB mRNA implying that CRR2 could also function in the stabilization of processed RNAs; however, future research is needed to confirm this interaction [The  to ndhB . Additiosystem I . A furth editing . Interes editing . Althougeraction ,50.Physcomitrella patens, later renamed to PpPPR_38, was initially shown to be involved in splicing of clpP and processing of the clpP–rps12 intergenic region resulting in elevated levels of precursor mRNAs [clpP mRNA and to the clpP–rps12 intergenic region in vitro. Studies with isolated chloroplast lysates suggested that binding of PpPPR_38 to the clpP–rps12 intergenic region stabilizes a predicted stem–loop structure that first provides a correct intergenic endonucleolytic processing site and then protects the cleaved clpP mRNA against 3′→5′ exoribonuclease activity [The PPR531-11 protein from or mRNAs . In a foactivity .RNA-Recognition Motifs (RRM)-containing, abundant stroma-localized proteins that associate with various ribosome-free mRNAs and pre-tRNAs [psbA, rbcL, petD, and rps14 as revealed by in vitro studies [Arabidopsis has a more specific function and was shown to be important for editing of different sites and stabilization of several but mainly ndhF mRNAs, whereas CP31B was assumed to support editing of specific CP31A-dependent sites [psbB, psbD, psaA/B, atpB, and ndhB to protect them from degradation under cold stress conditions [Arabidopsis CP33A protein is another example for the global role of cpRNPs in processing and stabilizing of multiple chloroplast RNAs [In general cpRNPs are divided into three different groups according to predicted structural features and phylogenetic considerations: I (cp28 and cp31), II (cp29A and cp29B), and III (cp33) . CpRNPs re-tRNAs ,83. Intere-tRNAs . Meanwhi studies . In addi studies . In cont studies . CP31A int sites . Furthernditions . The Araast RNAs .hcf145 mutant was initially found to specifically lack PSI [psaA–psaB–rps14 transcript encoding the two photosystem I core proteins PsaA and PsaB. Binding of HCF145 to the psaA 5′-UTR probably protects it from exoribonucleotytic attack [transcript-binding motif repeat (TMR) domains of approximately 70 amino acids which are responsible for specific binding to the psaA 5′-UTR. The TMR motifs are present rarely as single domains but often as multiple (up to ten) repeats in quite diverse proteins of unknown function in photosynthetic organisms including moss, red and green algae, and cyanobacteria [The seedling-lethal lack PSI . This coc attack . The HCFbacteria . The preArabidopsis proteins with unknown function and localization [Two additional N-terminal repeated regions of unknown function were found in the HCF145 protein that showed homology to the ubiquitously distributed SRPBCC  binding domains superfamily consisting of 11 Pfam families, including START domains, phosphatidylinositol transfer proteins, Bet_v_1, CalC related, CoxG, and polyketide cyclase related families . The strlization .psaA mRNA, although they do not contribute to the binding on their own. This points to a regulatory role of the SRPBCC domains in adjusting PSI levels [psaA RNA. Further studies will show whether a metabolism-dependent regulation of psaA–psaB–rps14 mRNA levels is important during development, greening and/or acclimation processes.Interestingly, the SRPBCC-related ligand-binding motifs support association to the 5′ I levels  and provprfB mutants described so far, in Arabidopsis lines lacking PrfB1 most plastid UGA stop codon-containing transcripts are unstable [C. reinhardtii, a PrfB-related gene is lacking in the nuclear genome and accordingly TGA stop codons are absent in their plastid genomes [prfB1 mutants. In either case, a hitherto unrecognized surveillance system efficiently removes all these nonfunctional transcripts. This demonstrates an advanced and more sophisticated regulatory level of plastid gene expression in comparison with known eubacterial systems.Different from eukaryotes harboring only one release factor (eRF) for ribosomal release at all three stop codons, eubacteria and organelles contain two release factors, PrfA (RF1) and PrfB (RF2), for termination of translation at UAG and UGA stop codons respectively. The UAA codon is recognized by both factors. PrfB1 and PrfB2 encode the only functional plastid and mitochondrial ribosomal RF2-related proteins in land plants respectively . In contunstable ,95. The  genomes . It is lInterestingly, a possible regulatory function of translational termination at UGA stop codons is reflected by a significant increase in the TGA content in land plant plastid genomes in comparison with those in algae . PresumaArabidopsis are able to terminate translation at UGA stop codons but are specifically affected in the accumulation of the cytochrome b6f complex due to the lack of petB transcripts encoding cytochrome b6. The petB gene is part of the primary pentacistronic psbB–psbT–psbH–petB–petD transcript encoding three proteins of PSII  and two proteins of the cytochrome b6f complex (PetB and PetD). Similar to most plastid polycistronic transcripts, numerous endonucleolytic processing events generate a wealth of overlapping RNA species, which are subjected to individual life times and translational control. Data revealed that petB transcripts that are cleaved at the 3′ end are efficiently generated but rapidly degraded in prfB3 mutants indicating that PrfB3 protects these RNAs from 3′→5′ exonucleolytic attacks. This is in agreement with preferential binding of PrfB3 to the petB 3′-UTR with respect to control transcripts [petB-containing precursor transcripts accumulate in prfB3 but petB is not translated. This indicates that binding of PrfB3 to the 3′ end could have an impact on elongation and/or initiation of translation at the 5′ end or that only processed monocistronic petB transcript are translational competent. Alternatively, a potential weak binding of PrfB3 also to the petB 5′-UTR is required for efficient initiation of translation [Land plant genomes surprisingly encode an additional eubacterial RF2-related protein, named PrfB3, which lost its two most important “SPF” and “GGQ” motifs of a release factor and thus the capability to terminate translation at UGA and UAA stop codons . Accordinscripts . Surprisnslation .petB mRNA levels [b6f amounts. Therefore, PrfB3 could well serve a regulatory function in vivo during acclimation processes by rate limiting levels of petB transcripts when changes in the amount of the cytochrome b6f complex are required. A similar regulatory role was assigned to the plastid PPR protein MCA1 in Chlamydomonas in determining levels of petA mRNA and accordingly its product cytochrome f [A drop in the expression of PrfB3 in partially complemented mutant lines correlated with a decrease in A levels . Most imchrome f .Arabidopsis mutation, which was characterized by a lack of some but not all oligo- and monocistronic psbH transcripts generated by processing of the pentacistronic psbB–psbT–psbH–petB–petD precursor message [psbH messages rather than processing is affected. PsbH encodes an essential 8 kDa phosphoprotein of PSII whose absence leads to a pronounced deficiency of PSII activity and protein levels in the hcf107 mutant [psbH in the nuclear genome, which targets the protein back to the chloroplast, revealed that HCF107 is exclusively required for the expression of psbH [C. reinhardtii that affects expression of psbB [The HCF107 gene was initially identified by high resolution mapping of the corresponding  message ,56. Dete7 mutant ,56. Ecto of psbH . This is of psbB .psbH start codon in agreement with the one repeat—one nucleotide model of PPR proteins [psbH transcripts lacking in hcf107. Expectedly, binding to its target protects the psbH RNA from 5′→3′ exonucleolytic attack in vitro [psbH 5′-UTR remodels the RNA structure, which may also allow ribosome binding and subsequent enhancement of translation, hinting to a dual role of the HCF107 protein. Obviously, PPRs and HATs can act in a similar and presumably analogous way to the recently identified family containing predicted superhelical and repeated RNA-binding TMR motifs in photosynthetic organisms [HCF107 encodes a half-a-tetratricopeptide (HAT) protein containing 11 repeats and no additional domains . It is mproteins  and accoin vitro . RNA strrganisms .Here, we summarized the characteristics of plastid RNA-binding proteins with a protective role in vascular plants. Whether the regulation of endonucleolytic activities, which were assumed to initiate degradation, is rate-limiting for mRNA decay still remains elusive. The RNA-binding proteins addressed here evolved mostly to hinder exonucleolytic attacks of tRNAs and translationally competent mRNAs, some of which were also shown to impact additional processing events and/or translation. Ribosome profiling was shown to partially overcome the challenge to investigate the role of the protectors in translation in particular using genetic approaches with low abundant RNA targets in the respective mutants ,100. NevpetB in Arabidopsis [b6f complex during stress and/or acclimation processes still needs to be shown.Differential expression of RNA protector genes under changing environmental conditions and the effects on their downstream targets speaks in favor of the idea that RNA degradation is mostly regulated at the RNA extremities. One example is provided by a comparable differential expression of AtprfB3 and its target bidopsis . WhetherpsaA stability and thus photosystem I amounts [The progression in mass spectrometric methods will prompt future studies on the metabolic impact and post-translational modifications of RNA protectors on the regulation of their activity, interaction with other hitherto unidentified association partners, and/or RNA affinity in a quantitative manner. Generally, these analyses remained poor, nevertheless the potential integrator HCF145 seems to be a promising candidate for such experiments. The two SRPBCC ligand binding domains of HCF145 offer an excellent platform for their metabolic control by the integration of external stimuli to finally fine-tune photosynthesis by regulating the effect of HCF145 on  amounts . Also, c amounts . Studies amounts ,4.Most protectors are plant specific congruent with the fast divergence of their targets at the 5′- and 3′-UTRs of plastid transcripts . Even ifChloroplast gene expression is predominantly governed on the level of post-transcriptional processes including RNA editing, methylation, polyadenylation, splicing, endo- and exonucleolytic digestion as well as translation.Generally, newly evolved and vascular plant-specific RNA-binding factors encoded in the nuclear genome are required for protecting specific chloroplast RNAs at exo- and endoribonuclease-sensitive sites reflecting a fast-evolving RNA metabolism congruent with the diverging RNA targets.Chloroplast RNA-binding protectors, usually found in high molecular weight complexes with largely unknown components, can act as RNA chaperones, occasionally affect other post-transcriptional processes, such as splicing, editing and translation, and contain a variety of different RNA recognition motifs, which often appear as multiple repeats.Interactions with other proteins, post-translational modifications of protectors, metabolic pathways, and presumably noncoding RNAs may have an important impact on the characteristics of RNA protectors, which are required for normal plant growth and development as well as diverse stress responses and acclimation processes.


Journal Club and to highlight some of the most interesting papers published recently. We hope to help you keep aware of non-coding RNA research outside of your area. The Non-Coding RNA Scientific Board wishes you an exciting and fruitful read.We are glad to share with you our third Highlight by Claire Francastel and Florent Hubéi.e., ribosome profiling coupled to high throughput sequencing) first developed by Nick Ingolia and Jonathan Weissman. In their recent review, Muhammad Ali Mumtaz and Juan Pablo Couso provide a thorough description of the method and the type of data that one can expect through this approach [i.e., predict elongation rate and pausing. Unexpectedly, about half of lncRNAs described so far are found associated with polysomes in mouse embryonic stem cells. Whether this association leads to the production of tiny peptides remains to be seen. However, the main conclusion of the authors and related work, “The differences between coding and noncoding RNA are likely to be blurred”, tears down one more, and maybe the ultimate, principle that distinguishes coding mRNA from “non-protein coding” ncRNAs.Over the last few years, a growing number of studies have described neglected small open reading frames sORF; <100aa) hidden in long non-coding RNAs (lncRNAs), using ribosome footprinting  emerged a few years ago as functionally important molecules involved in nearly all physiological and pathological processes of the cell. Numerous classes of sncRNAs have been reported that include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) of course, but also many others like microRNAs (miRNAs) and small nucleolar (snoRNAs), the best known among them. For each class, many databases were built, some of which are now recognized references databases; this is the case for miRBase.org, which is the reference searchable database for annotated miRNAs. However, these databases are not complete since, for example, a few miRNAs annotated in UCSC were still not deposited in miRBase. Moreover, as many databases of classes of sncRNA of interest have to be assembled and curated for a given study. This drawback has now been fixed with the DASHR database from Leung et al. . Their dHighlight by Claire Francastel and Florent HubéA class of small non-coding RNAs (sncRNAs), derived from mature or precursor transfer RNAs (tRNAs), are 14 to 32 nt-long single-stranded RNAs, clearly distinct from canonical microRNAs (miRNAs). There is growing evidence that these tRFs are not mere by-products of tRNAs degradation, but, instead, represent functional sncRNAs. Some tRFs are constitutively generated while others are produced in stress conditions. They appear to account for miRNAs reads in small RNA-seq data. The paper by Shigematsu and Kirino represents the first review of its kind on tRFs biogenesis and function in various organisms [Highlight by Gaetano SantulliDrosophila larvae [In a very elegant paper published in Science, Joao Picao-Osorio and colleagues demonstrated that specific microRNAs (miRNA) are functionally involved in the control of behavior . A mutata larvae . Small RSince Hox gene post-transcriptional regulation has been found to be involved in self-righting control, other RNA-based regulatory processes affecting Hox gene expression may also influence neural output. These findings will most likely prompt researchers toward the study of miRNAs involved in the control of behavior in other organisms, including humans.Highlight by Ioana Berindan NeagoeFischer and the group at Weill Cornell Medical College , demonst


Retinoblastoma is rare tumor of the retina caused by the homozygous loss of the Retinoblastoma 1 tumor suppressor gene (RB1). Loss of the RB1 protein, pRB, results in de-regulated activity of the E2F transcription factors, chromatin changes and developmental defects leading to tumor development. Extensive microarray profiles of these tumors have enabled the identification of genes sensitive to pRB disruption, however, this technology has a number of limitations in the RNA profiles that they generate. The advent of RNA-sequencing has enabled the global profiling of all of the RNA within the cell including both coding and non-coding features and the detection of aberrant RNA processing events. In this perspective, we focus on discussing how RNA-sequencing of rare Retinoblastoma tumors will build on existing data and open up new area’s to improve our understanding of the biology of these tumors. In particular, we discuss how the RB-research field may be to use this data to determine how RB1 loss results in the expression of; non-coding RNAs, causes aberrant RNA processing events and how a deeper analysis of metabolic RNA changes can be utilized to model tumor specific shifts in metabolism. Each section discusses new opportunities and challenges associated with these types of analyses and aims to provide an honest assessment of how understanding these different processes may contribute to the treatment of Retinoblastoma. Rb1: gene, pRB: protein) gene and the E2 promoter binding Factors (E2Fs) function to control both of these pathways  cells as the major cells of origin of Retinoblastoma . High reFigure 1).Pioneering studies into the transcriptome of Retinoblastoma tumors using microarrays have provided critical foundations for our understanding of the disease. The advent of new and unbiased profiling of the RNA content of cells by RNA-sequencing (RNA-seq) significantly increases the resolution of the RNA measurements and can be expanded to include non-coding expression and detection of rare RNA fusion products. RNA-seq provides enhanced transcriptomic profiling by directly sequencing the RNA or DNA copies of the RNA and comparing it to the genome rather than relying on probe detection on traditional microarrays. This technology therefore circumvents the need to test RNA on pre-determined gene sets and can interrogate the entire transcriptome. RNA-seq platforms can measure all the RNA of cells, however, require the removal of very abundant RNA species within the cells that do not contribute to the transcriptome including ribosomal RNA (rRNA) and transfer RNA (tRNA). This critical experimental step depletes these specific RNA species from the cellular pool of RNA before library preparation and increases the overall coverage and depth of RNA-seq reads on the remaining genes. Currently RNA-seq datasets of Retinoblastoma tumors and normal controls have not been published, however, several independent teams are known to be working to generate these exciting datasets. In this perspective, we discuss three exciting opportunities that RNA-seq datasets of Retinoblastoma tumor may provide to the research community . E2F1-3 are potent inducers of transcription and in particular genes required for cell cycle progression and apoptosis, however, very little is known about how these factors control LINC-RNA expression. Preliminary studies from cell lines mapping activator E2F-mediated regulation of LINCs have suggested that a significant number of these RNAs may show transcriptional changes upon RB-loss via both direct and in-direct mechanisms . In addiin vitro or in model organisms changes the LINC expression profile is limited, determining how RB1 homozygote mutation affects LINC levels in a complex and developmentally disorganized Retinoblastoma tumor represents a real challenge. With the development of RNA-sequencing that unbiasedly measures the levels of RNA in cells without the need to purify polyadenylated mRNAs or have probes designed against the transcriptome we can now, for the first time, interrogate the entire transcriptome of these tumors. This new approach will enable the development of Retinoblastoma specific LINC-RNA profiles and may generate clinically predictive biomarkers for identifying patients likely to develop secondary tumors. However, significant technical challenges have arisen in other tumor types when profiling LINC-RNAs. These are generally due to the lower expression levels of most LINC-RNAs compared to coding mRNAs and the high variability between patients. RNA-seq reads at the lower end of the runs tend to have greater signal to noise levels than better expressed mRNAs which can make generating predictive LINC profiles from small numbers of tumors difficult. This is a potential issue in Retinoblastoma studies as these tumors are rare and both normal control and tumor cohort size tends to be smaller than for more common adult malignancies.Given that our understanding of how RB1 loss Figure 2). Profiling Retinoblastoma by RNA-seq technology will help fill some of the gaps in our knowledge and provide new insights into how RB1-loss may change the LINC-RNA transcriptome of these aggressive tumors but also to those in more genetically complex adult tumors.Although there are currently significant gaps in our understanding of how RB1 and the E2F pathway modulate the expression of the LINC-RNA transcriptome in cells and in tumors there seems to be little doubt that expanding our knowledge about how RB1-loss changes the entire transcriptome is an exciting and clinically relevant question . Such events would normally be excluded or undetectable with traditional microarray technology. Finding and mapping these rare events is an area of growing interest as the aberrant proteins are potential neo-antigens which can be recognized by the patients’ immune system. A number of groups have highlighted the utility of these “abnormal” products for the clearance of tumors, although as yet not in Retinoblastoma, to immunotherapy. This is an emerging clinical avenue and in particular could have significant advantages in the treatment of Retinoblastoma as it would reduce the exposure of pediatric patients to developmentally harmful chemotoxic treatments. The detection and mapping of these events is possible with deep RNA-seq coverage and may provide insights into how splicing and/or RNA metabolism is changed in Retinoblastoma tumors.In addition to the documentation of LINC-RNA and other non-coding RNA changes caused by RB1 loss, RNA-seq also enables the measurement and identification of novel or fusion RNAs in samples. The pRB protein has important roles in modulating genomic integrity and stability and loss of pRB has been shown to contribute to accelerated genome manipulations and enhanced drug sensitivity. RNA-seq of Retinoblastoma tumors may also enable the identification of novel RNA species caused by genomic rearrangements or aberrant RNA processing events . This approach has previously been employed to assay model organisms, tissue and drug treatment network maps . One limMicroarray profiling of Retinoblastoma tumors has been instrumental in identifying the aberrant pathways driving the growth and invasion of the tumors. These approaches have really built an immense foundation that has enabled the Retinoblastoma research field to test and develop new ways to therapeutically target these tumors. This technology, that has served us so well, has a number of limitations that can be largely circumvented by the use of RNA-sequencing. In particular, RNA-seq measures both coding and non-coding RNAs changes which may be invaluable to understanding the biology of these tumors but also in helping to stratify patients into different treatment outcome or relapse groups. Collectively, this tool enables us to interrogate the biology sustaining Retinoblastoma tumor growth and may led to discovery of new therapeutic approaches to treat these patients.The protocol was approved by institutional review board on Ethical practices for research at Sankara Nethralaya.SE and WM contributed to the conception, preparation, and revision of the manuscript. SR built the figures. All of the authors approved and agreed on the manuscript.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.


Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa. This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management.Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales. Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator. Apis mellifera  and Deformed wing virus (DWV) (Black queen cell virus (BQCV) and Acute bee paralysis virus (ABPV)  , 23, ands (ABPV) . More res (ABPV) , which ls (ABPV) . LSVs ars (ABPV) .Varroa destructor  pathway , 37, 38.Varroa parasites but do not appear to suffer any negative consequences , Stellenbosch and Robben Island (South Africa), and Vava'u and Tongatapu islands (Kingdom of Tonga)  . The colof mites . Colonieto mites . The honmain low . In the f Vava'u . We syntf Vava'u . We asse Trinity  and compVarroa-resistant populations. Contigs for DWV, BQCV, and sacbrood virus (SBV) were present in all three locations. ABPV was present in one colony from Robben Island, and LSV was present in one colony each from The Netherlands and Tonga and in two colonies from South Africa  found in Drosophila  (Apis mellifera), followed by the name or category of virus and number if more than 1 .Next, we examined contigs that showed similarity to previously characterized virus sequences from positive-sense, negative-sense, and double-stranded RNA genomes available in GenBank. We found genomic evidence of seven previously undescribed viruses in the honey bee transcriptomes, including four negative-sense and three positive-sense RNA viruses  and 3. Oavirales , and twoaviridae . The throsophila , and oneus (DCV)  to E. FoOrthomyxoviridae family (22% identity), which suggests that this glycoprotein gene may be acquired through an inter-virus-family horizontal transfer event  has a 14,613-nt genome with a prototypic rhabdovirus structure  correspoer event . Intereser event  and 3, wer event . ARV-2 wer event  and 3. Ter event . The RdRBunyaviridae are negative-sense RNA viruses with three genome segments , each containing a separate ORF . Ph. PhBunyatebrates  and placFlaviviridae (Flaviviridae placed AFV in a clade of other recently discovered flavi-like viruses with large genomes (Drosophila species (Takaungu virus and Hermitage virus) (Apis mellifera flavivirus (AFV) was identified in one colony from Robben Island. The 20,414-nt positive-sense RNA genome contains a single ORF of 6,615 aa . BLAST siviridae . The phy genomes   .Cricket paralysis virus (CrPV), Drosophila C virus, and Anopheles C virus (We identified a novel dicistrovirus from one colony at the Amsterdam Water Dunes (Apis mellifera dicistrovirus [ADV]). The 9,126-nt genome contains two ORFs encoding the replication enzyme polypeptide and the capsid proteins, respectively, which is typical of dicistroviruses . The pol C virus .Drosophila species  and Sanger sequencing using primers spanning the neighboring contigs. The resulting Apis mellifera Nora virus (ANV) partial genome sequence is 10,091 nt long and covers the entire replicase, although it is missing the first ORF at the 5′ end of a typical Nora virus genome  53). In. In53).  species .One way of confirming that a putative virus genuinely infects the host from which it is sampled is the presence of an antiviral immune response. In insects, likely candidates are the small RNA pathways that are utilized in viral defense , 40, 59.A. mellifera samples: two from the Amsterdam Water Dunes and two from Robben Island. For each of these geographic locations, we used PCR to screen for individuals that were positive for ARV-1 and used one individual that tested positive for ARV-1 (Amsterdam Water Dunes positive [AWD+] and Robben Island positive [RI+]) and one individual that tested negative (Amsterdam Water Dunes negative [AWD−] and Robben Island negative [RI−]). The RI+ sample also tested positive for ARV-2. The four libraries were subjected to 50-bp single-end sequencing, resulting in between 10 million and 18 million reads per sample.We generated small RNA libraries from the abdomens of four Apis mellifera genome and then aligned the unmapped reads to the ARV-1 and ARV-2 genomes  starting at the dsRNA termini and fading with increasing distance from the termini, with a characteristic 2- to 3-nt overhang . We lookP < 0.01 for both by a chi-squared test)   and H.Taken together, our data suggest that the ARV-1 and -2 small RNAs have been generated by Dicer acting on a double-stranded RNA replication intermediate and that the small RNAs are subsequently bound by Argonaute proteins, indicating that the bees have an active antiviral immune response against ARV-1 and ARV-2.V. destructor mites collected from A. mellifera individuals from Robben Island and performed Illumina 50-bp single-end sequencing, resulting in 18 million to 20 million reads per sample , no 5′-nucleotide bias for antisense reads for ARV-1 (not significant [NS] by a chi-squared test), and no 5′-nucleotide bias for sense or antisense reads mapping to ARV-2 (NS by a chi-squared test)   and J. TAedes aegypti  ping-pong amplification pathway, as this pathway has been implicated in viral defense in  aegypti , 63. The aegypti . We geneThe small RNA profiles in mites do not mimic those in honey bees, indicating that the mite reads do not come from ingested honey bee material. Thus, the high abundance of reads in the mites  and the Varroa-resistant or Varroa-free honey bee populations from three locations in Europe, Africa, and the Pacific. We present genomic evidence of seven new RNA viruses, including three novel positive-sense and four novel negative-sense viruses. Our study therefore increases the number of known honey bee viruses from 24 to 31. ARV-1 and -2 were found in three geographically distinct populations. Using small RNA sequencing, we show that honey bees exhibit classic Dicer-mediated siRNA profiles, suggesting an active bee immune response. We also report the first analysis of small RNAs in Varroa destructor mites and show that ARV-1 and -2 are present in these mites, although the small RNA profile is distinctly different from that for honey bees.We describe a diverse set of new viruses in Osmia cornuta, including one virus from the order Mononegavirales and one from the family Bunyaviridae, indicating that viral diversity in other hymenopteran species extends to negative-sense viruses  are related to viruses known to be present in insects . Our fin viruses . One of pmorLBV1 . LepmorLra flies . Interesa passim . We therThe small RNA patterns of the rhabdoviruses in honey bees show classical Dicer-mediated degradation profiles, providing strong evidence that ARV-1 and -2 enter the cells of bees and begin to replicate. During viral replication, a double-stranded RNA replication intermediate is formed, which can be recognized by the RNAi machinery and chopped by Dicer. The resulting small RNAs become part of an antiviral immune response . This stVarroa, the strong bias for 24-nt antisense small RNAs and the lack of phasing suggest that replicating dsRNA is not a template for Dicer. Internal secondary structures within a negative-sense virus genome can provide dsRNA templates for Dicer . In July 2014, 10 colonies of A. mellifera bees from an apiary at the Amsterdam Water Dunes, The Netherlands, where Varroa has been present since the 1980s and where natural selection for Varroa-resistant colonies has been ongoing since 2008, were sampled  and transported at room temperature prior to storage at −80°C until processing. In Africa and The Netherlands, a minimum of 10 adult worker honey bees were sampled per colony, frozen immediately on dry ice, and stored at −80°C until processing.Honey bee colonies and mites from Europe, Africa, and the Pacific were sampled in 2013 to 2015. Seven  5 years . On Robb sampled . In OctoThorax and abdomen tissues of five adult bees from each colony (thorax only from Tonga) were homogenized in TRIzol reagent (Thermo Fisher), and total RNA was extracted according to the manufacturer's protocol. RNA from each individual was diluted to 200 ng/μl, and 2.65 ng RNA from each of the five individuals from each colony was pooled prior to DNase treatment (Ambion), followed by column purification . Total RNA was transported to the Australian Genome Research Facility (AGRF) on dry ice. Sample RNA integrity was confirmed by using a Bioanalyzer (Agilent). RNA was subjected to a ribosome depletion step (Ribo-Zero-Gold Human/Mouse/Rat) prior to the preparation of Illumina TruSeq Stranded Total RNA paired-end libraries according to the manufacturer's instructions. Libraries were run on an Illumina HiSeq2000 100-bp paired-end sequencing system for a total data yield of 4 to 9 Gb per sample.de novo by using Trinity . We also confirmed the arrangement of the ARV-2 G and L proteins and the putative inter-virus-family horizontal transfer event, using PCR for amplification across the two ORFs and Sanger sequencing for confirmation.We used Bowtie2  to map rVarroa mites was used to generate a small RNA library by using a TrueSeq small RNA kit (Illumina) according to the manufacturer's protocol. Samples were barcoded appropriately for pooling on an Illumina HiSeq2000 instrument, with 50-bp single-end sequencing.One microgram of total RNA (prepared as described above) from individual bees and A. mellifera genome, allowing for up to two mismatches (length fraction of 1 and similarity fraction of 0.9) due to the divergence between honey bee strains and the reference genome. The unmapped reads were subsequently mapped to ARV-1 and ARV-2 with the same stringency settings. The Amsterdam Water Dunes and Robben Island samples were mapped to the consensus viral genome generated by RNA sequencing (see above) from their geographic location. For further small RNA analysis , the mapped reads were exported as BAM files, indexed by using Samtools . The reads were first mapped to the Samtools , and theSamtools  and custx + 22 nucleotides along the sequence (iP)  using custom R scripts and then divided this value by the number of remaining out-of-phase reads (U). Phasing scores were calculated for each phase cycle position in the 22-nt window over 8 cycles, as the signal is expected to degrade over time due to imprecision in the cut length. The relative accumulation of reads at a particular phase cycle position will give a high phasing score. For the honey bee data, we specifically looked for a phase cycle of 22, since this was the predominant size of the small RNAs. For the Varroa data, we analyzed four different phase cycle lengths  using the following equation:i is the phasing cycle, iP is the number of small RNA reads at a given phase cycle position, U is the number of small RNA reads within the phase cycle not at the phase cycle position (out of phase), and n is the number of phase cycle positions occupied by at least one small RNA within the 8 cycles.To determine whether the small RNAs produced from the viral genomes showed evidence of Dicer phasing, we adapted an algorithm designed for detecting phased RNAs in plants . The logKY354230 to KY354234 and KY354236 to KY354244. Annotated NCBI protein sequence accession numbers are ARO50020 to ARO50067. Raw sequence data have been deposited in the Sequence Read Archive (SRA) (accession number SRP095071) (BioProject accession number PRJNA357165 and BioSample accession numbers SAMN06140203 to SAMN06140219).All virus genome sequences generated in this study have been deposited in the GenBank database under accession numbers


Motivation: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not.Results: We present here V’DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V’DJer’s ability to accurately reconstruct BCR repertoires from short read mRNA-seq data.Availability and Implementation: V’DJer is implemented in C/C ++, freely available for academic use and can be downloaded from Github: https://github.com/mozack/vdjerContact:benjamin_vincent@med.unc.edu or parkerjs@email.unc.eduSupplementary information:Supplementary data are available at Bioinformatics online. This diversity of targeting receptors is crucial for immune defense against infectious pathogens and has implications for understanding autoimmunity, immunodeficiency syndromes and the anti-tumor immune response to malignant diseases.T-cells and B-cells compose the adaptive immune system and bear highly specific cell surface receptors that allow them to recognize antigenic targets. Massive diversity at the adaptive immune receptor loci is generated by the process of V-J and V-D-J recombination during cell development, with a theoretical number of unique receptors for each class estimated at greater than 10Adaptive immune receptor repertoire analysis was initially performed by evaluating differences in sequence length of the primary antigen binding region, a process known as spectratyping  file of mapped mRNA-seq short reads as input. V’DJer then performs:V’DJer outputs a fasta file containing BCR contigs and a Sequence Alignment/Map (SAM) file containing reads mapped to those contigs . This ouSupplementary Note 1) as follows:Reads mapping to any functional locus specific to the IG chain of interest are extracted.Mapped reads containing a 15-mer in common with the IG chain’s functional germline V, D or J segments are extracted.All unmapped reads are extracted.In all cases, a read is always extracted along with its paired read.V’DJer extracts reads from a STAR . Traversal of a path through the graph continues until one of three conditions is met:Candidate contigs are generated from paths reaching an appropriate length. The Samtools API  sequence. Sixteen base long anchors are extracted from near the 3′ end of germline V segments and 5′ end of J segments. Candidate contigs are searched for sequence showing homology to at least one V and one J anchor (maximum of four mismatches). If the V and J anchors are within a reasonable distance of one another, the contig is searched for amino acids known to be highly conserved in CDR3. Cysteine is required to appear on the 5′ end of the CDR3 region while Tryptophan (IgH) or Phenylalanine (IgK/IgL) must appear on the 3′ end. These amino acids must be in frame and within expected CDR3 length distance from one another. If a contig passes these initial CDR3 identification criteria, the originally extracted reads are mapped to the contig using a hashing approach. All reads are stored in a hash table and the contig is examined from beginning to end looking for perfect read matches. If both members of a read pair map to a contig in the correct orientation and within a reasonable insert length, they are considered mapped. While computationally efficient, this approach does not currently allow for sequencing errors. The relatively exhaustive graph traversal that V’DJer employs would ordinarily result in a potentially high number of false positive paths through the graph. To resolve this, candidate contigs are required to have sufficient read depth and complexity of coverage to pass the evaluation step. Those contigs that do pass are output along with read alignments to those contigs.The V’DJer output is a fasta file containing assembled contigs spanning most of the V(D)J region as well as a portion of the constant region . Additionally, a SAM file containing reads aligned to the assembled contigs is output. We use RSEM to quantify the assembled transcripts. VQuest is used to gather additional information about the assembled contigs including V and J segment identification as well as V region identity which we use as a proxy for mutational load. BCR isotypes are identified by mapping the trailing 48 bases of each contig (constant region sequence) to hg38 using STAR and identifying the constant region segment using the resultant coordinates. Contigs sharing an identical CDR3 (amino acids), V gene segment, J gene segment and isotype are clustered for downstream analysis.Supplementary Note 2). The relative abundance of V’DJer called BCR sequences was then estimated by RSEM (2 = 0.995) . For the majority of combinations (1015 of 1410), 100% of simulated clonotypes were detected, and for all combinations at least 70% of simulated clonotypes were detected (Supplementary Table 1).We initially tested V’DJer’s ability to accurately assemble BCR V(D)J sequences by analyzing simulated repertoires of IgH clonotypes (unique sequence arising from VDJ recombination and somatic hypermutation) from which short paired reads were generated at average depths varying from 25× to 500×. When run in standard mode, V’DJer achieved greater than 90% sensitivity at an average clonotype depth of 50× or greater with zero false positives . Running= 0.995) . FinallySupplementary Note 3). In parallel with IgH amplicon sequencing, V’DJer was run on 2×50 paired-end bulk mRNA sequencing data from the same samples. For this evaluation, assembled contigs encompassing at least one merged MiSeq read were considered validated. Of those V’DJer assembled contigs comprising at least 1% of the IgH repertoire as computed by RSEM, 85.5% were validated by the MiSeq sequencing (We further assessed V’DJer’s performance by comparing to targeted IgH amplicon sequencing of breast cancer derived RNA samples. MiSeq 2×250 paired end sequencing was used for this comparison with primers originating from framework region 2 of various IgH variable (V) gene segments as well as a joining (J) gene segment used in the IgH repertoire amplifications  (2 = 0.949) (P = 1.07 × 10−53) (p = 0.253). .We applied V’DJer to 473 Melanoma samples from TCGA , followe= 0.851) . This re= 0.949) . For tho= 0.949) . CDR3 le= 0.949)  were in = 0.949) , with Ig× 10−53) . No indiMeasurements of diversity can include assessments of richness (the number of distinct members of the repertoire) and evenness (variance of abundance of clonotypes within the repertoire). Here, we use Pielou’s evenness index as a measure of BCR repertoire diversity within each sample calculated as the Shannon entropy divided by the log of the number of clonotypes.This measure of diversity serves as an indicator of possible clonal selection and expansion. A sample with a single or small number of clonotypes expressed at levels much higher than other clonotypes in the sample would be considered to have low evenness, while a sample with clones of similar abundance would have high evenness . We asseMean V’DJer run time for the 473 sample TCGA Melanoma cohort on the IgH chain was <3 h running on 8 core servers while mean peak RAM usage was <12GB. Samples containing high BCR abundance and diversity used considerably more compute time and RAM, in some cases more than a day of processing and >32GB of RAM.We present here V’DJer, software for inference of BCR repertoires using short-read RNA sequencing data. A major advantage of this approach is the capacity to capture expressed BCR loci in a standard RNA-seq assay. This is especially important in application to clinical samples where nucleic acid template may be limiting. In contrast to assays that depend on PCR amplification, V’DJer is not limited to evaluating only those sequences that can be successfully primed. V’DJer does not require FACS sorting of B-cells prior to analysis; rather it performs well when analyzing complex RNA template mixtures derived from the bulk tumor immune microenvironment.Application of V’DJer is primarily limited by read coverage of the clones of interest. In the setting of high coverage, assembly sensitivity and specificity are high as well; however in areas of low coverage sensitivity lessens. Thus in the context of a tumor immune infiltrate, V’DJer is best suited for reconstruction of dominant and subdominant clones. The algorithm allows for wide latitude in choosing parameters for optimal performance given the expected coverage in the sample; however there is a lower bound on coverage for recovering a given clonotype in the assembly. Functional BCRs are protein multimers, comprising heavy chain and kappa or lambda light chain pairing. Recently, multiple methods have been developed to capture paired heavy/light chain information  (Other assembly-based methods have been used to analyze adaptive immune receptor repertoires. The first of these was iSSAKE, which assembled short read mRNA-seq data derived from sequencing 5′ RACE products to reconstruct TCRβ repertoires .Adaptive immune receptor repertoire profiling in general is an important analytical tool for translational cancer biology. Our group and others have shown that B-cell lineage gene expression signatures are strongly prognostic in multiple solid tumor types (


The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples  is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5′ tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5′ tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5′ tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression and play important roles in essential physiological and pathological processes3Several measurement technologies are available for assessing relative miRNA abundance: microarray, RT-qPCR and massively parallel sequencing. With differences in reproducibility, accuracy, sensitivity and specificity, the method of choice is strongly dependent on the specific research question789In serum, certain tRNA-derived fragments called 5′ tRNA halves appear to be abundantly present, consuming the majority of sequencing reads1112131516171819In this study, we evaluated two complementary approaches for selective depletion of 5′ tRNA halves from murine serum derived RNA based on bead capturing and RNase H cleavage, respectively. Using bead-based depletion, we were able to reduce the 5′ tRNA halves isotypes by more than 95%. This enabled us to increase the number of reads mapping to miRNAs by 6-fold, with 60% more unique miRNAs detected and importantly, with no differential effects on the constitution of the remaining miRNA population. To demonstrate the benefit of performing a 5′ tRNA half depletion, we investigated differentially expressed miRNAs between tumor-free mice and mice carrying orthotopic xenografts. In line with a higher detection rate, the selective depletion of 5′ tRNA halves increased the detectable amount of differentially expressed miRNAs by a factor of three compared to the same non-depleted RNA samples.First, we evaluated the effect of the small RNA library preparation method on the 5′ tRNA-halves abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits. RNA isolated from 100 μl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.Even though all libraries were sequenced at the same sequencing depth, we observed a clear difference in the number of mapped reads between the different library prep kits. Using the NEBNext kit we obtained between 17.1 and 26.4 million mapped reads, with the TruSeq kit between 10.0 and 12.5 million mapped reads and with the TailorMix kit between 5.3 and 5.5 million mapped reads. Annotation analysis of the mapped sequencing reads revealed that all libraries contained high amounts of reads mapping to tRNA genes, irrespective of library preparation method . We founInvestigation of the different types of 5′ tRNA halves present in the libraries revealed that the majority of 5′ tRNA half reads corresponded to just a small subset of tRNAs types. The most abundantly present tRNA types are those with glutamine, glycine, valine and histidine isoacceptors. The relative percentages of these tRNA types vary between the library preparation kits, but their total relative contribution is similar and close to 95% of all tRNA reads .Next, we evaluated two complementary protocols for 5′ tRNA half depletion for the four tRNA types that we found to be most dominantly present in murine serum: glutamine, glycine, valine and histidine.For each tRNA half type, different isoacceptors were detected. Comparison of the sequences of the different isotypes showed that it was possible to effectively deplete these fragments using only a limited number of probes for each tRNA type. We designed DNA probes complementary to the most abundantly detected sequences of the four most abundant tRNA half types. In total we designed five probes for depletion of the four most abundant tRNA halves. To interrogate for possible off-target effects, probe sequences were queried against the human and mouse miRNome to identify miRNAs that show perfect complementarity with the probes . In totaTwo complementary approaches were evaluated for their efficacy in depleting 5′ tRNA-halves . In a fiBoth protocols were evaluated on RNA isolated from a serum pool that was collected from healthy SVj mice by cardiac puncture. We assessed the 5′ tRNA half depletion efficiency using RT-qPCR and compared tRNA-half levels between depleted and non-depleted samples . For botTo assess whether the depletion protocols could affect miRNA expression values, we determined the abundance levels of mmu-miR-16-5p , a miRNA that is reported to be highly and stably expressed in mouse serum, in depleted and control samplesTo evaluate the efficacy of the described protocols on small RNA sequencing, we performed the two 5′ tRNA halves depletion protocols on total RNA isolated from a serum pool collected from healthy mice. Each RNA sample was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control. The experiment was performed in triplicate. Upon depletion, libraries were prepared using TruSeq Small RNA library preparation kit (given the higher number of mapped reads and miRNA reads as compared to the NEBNext and TailorMix kits). Samples were sequenced as described above.6, 1.77 × 105 and 5.46 × 106 for control, RNase H-depleted and beads-depleted samples, respectively. Comparison of the relative abundance of the different RNA species revealed a pronounced reduction in the fraction of detected 5′ tRNA halves in depleted samples as compared to non-depleted samples  in depleted samples . Of the Low RNA concentration due to its low abundance in serum poses a critical obstacle for the exact quantification of serum-derived miRNAs, especially for sequencing-based technologies. There have been several efforts to improve discrete steps of the miRNA quantification process in order to enhance the efficacy of miRNA detection in these types of samples. As such, improvement of pre-analytical parameters such as storage conditions and serum preparation, optimization of the RNA extraction process and advancements in library preparation protocols were found to improve miRNA detection performance25262728In this study we optimized and compared two protocols for the selective depletion of 5′ tRNA halves in RNA isolated from murine serum samples of which only one protocol was successful in targeted depletion of 5′ tRNAs halves. Using a protocol consisting of target capture by biotinylated DNA probes followed by immobilization using magnetic streptavidin beads, we were able to successfully deplete up to 99% of the targeted 5′ tRNA halves in RNA samples. Depletion of 5′ tRNA halves in small RNA libraries resulted in a more than 6-fold increase in mapped miRNA reads and 60% more detected mature miRNAs when performing small RNA sequencing on murine serum samples. Importantly, we observed no considerable effects on the relative expression values of miRNAs. Moreover, using 5′ tRNA halves depletion we were able to detect 3 times more differentially expressed miRNAs when comparing serum from mice carrying orthotopically engrafted tumors with serum from healthy control mice. We detected 34 differentially expressed miRNAs of which six are unique to humans, 13 unique to mice and 15 have complete conservation between the two species. Especially for such applications, in which the miRNA sequence itself can carry information about its cellular origin, sequencing is very well suited because of its exquisite specificity when strict mapping is applied. In contrast, RNase H-based depletion did not prove highly effective. It could be that cleaved tRNA half fragments are still being processed during library preparation as RNAse H can yield polished, easily ligated RNA ends.It is straightforward to further increase the efficacy of 5′ tRNA halves depletion by including additional capture probes that target other 5′ tRNA isoacceptors not targeted in this study. It has been reported that the expression of circulating 5′ tRNA halves derived from specific tRNA isoacceptors can change as a result of experimental conditions like aging and calorie restrictionHealthy male and female 129/SVj mice, with ages varying between 2 to 9 months, were sedated using tribromoethanol at a dose of 250 mg/kg injected intraperitoneally. 500 μl to 1 ml of blood per mouse was collected in Safe-Lock micro test tubes (Eppendorf) by cardiac puncture using a 0.45 × 12 mm needle after which mice were sacrificed by cervical dislocation. For NCr nude mice (Taconic Farms), 100 μl blood was collected by puncture of the jugular vein using a 4 mm lancet and collected in a BD Vacutainer collection tube with a gel separator (BD). All blood samples clotted at room temperature for 45 min. Clotted blood samples from healthy SVj mice were centrifuged at 1900 g for 10 min at 4 °C using a swinging bucket rotor. The supernatant, containing the serum, was collected and centrifuged again at 16000 g for 10 min at 4 °C in a fixed angle rotor to pellet cellular nucleic acids attached to cell debris. The supernatant was collected and stored at −80 °C. Clotted blood samples of nude mice were centrifuged at 2000 g for 15 minutes at 4 °C in a fixed angle rotor. The supernatant was collected and stored at −80 °C. For all serum samples, the degree of hemolysis was determined by measuring levels of free hemoglobin by spectral analysis. Absorbance peaks at 414, 541 and 576 nm are indicative of free hemoglobin6 human SH-SY5Y neuroblastoma cells were surgically implanted in beneath the renal capsule. This model closely resembles the growth characteristics of primary neuroblastoma arising from the para-adrenal location in humansOrthotopic neuroblastoma xenografts were generated in four to six week old female athymic immunodeficient NCr nude mice as previously describedRNA was isolated using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer’s instructions. For the final experiment, 50, 100 or 200 μl of serum was used as input and total RNA was eluted in 12 μl of RNAse-free water.RNA used for the evaluation of the different library preparation kits was isolated from 100 μl of serum collected from individual SVj mice and eluted in 12 μl of RNAse-free water. For RNA isolation of samples used for the assessment of effective tRNA depletion by sequencing, serum samples from 3 individual SVj mice were pooled and 200 μl aliquots of the serum pool were used as input for RNA isolation and total RNA was eluted in 12 μl of RNAse-free water. After elution from the miRNeasy column, RNA samples used for the assessment of tRNA depletion by sequencing were diluted to a final volume of 38 μl and distributed in 12 μl aliquots. RNA used for differential expression analysis was isolated from 50 μl of serum that was collected from Ncr nude mice and eluted in 12 μl of RNAse-free water.5′ biotinylated DNA probes were designed for four 5′ tRNA halves species  and ordeFor tRNA halves depletion using beads, 12 μl of RNA, 15 μl of 2x hybridization buffer , 1 μl ofFor tRNA halves depletion using RNAse H, 2 μl of 7.5x hybridization buffer  was combFor reverse transcription we used the miScript II RT kit (Qiagen) with HiFlex buffer according to manufacturer’s instructions but with 1.5 μl of input RNA. After reverse transcription, the resulting cDNA was diluted 22-fold by adding 210 μl nuclease-free water to 10 μl of RT product.For detection of tRNA-halves by RT-qPCR the miScript SYBR green PCR kit (Qiagen) was used with custom designed primer assays . The RT-For Small RNA library preparation we used the TailorMix miRNA sample preparation kit v2 (Seqmatic), the NEBNext Multiplex Small RNA library prep kit (New England Biolabs) or the TruSeq small RNA library preparation kit v2 (Illumina) following manufacturer’s instructions with small modifications . AdaptorFor RT-qPCR quantification of sequencing libraries, 1 μl of EtOH purified library was diluted 1:100 000. 2.5 μl of SsoAdvanced universal SYBR green supermix (Bio-Rad Laboratories) and 0.25 μl of each primer 5 μM;  were comFor the quantification of small RNAs Biogazelle’s dedicated small RNA seq pipeline was used (part of Cobra), as publically available alternatives we advice the use of miRDeep2 and miRExpress. Adaptor trimming was performed using Cutadapt v1.8.1, reads shorter than 15 bp and those in which no adaptor was found, were discarded. For quality control the FASTX-Toolkit (v0.0.14) was used, a minimum quality score of 20 in at least 80% of bases was applied as a cutoff. The reads were mapped with Bowtie (v1.1.2) without allowing for mismatches. Mapped reads were annotated by matching genomic coordinates of each read with genomic locations of miRNAs  and other small RNAs  and Ensembl, v84). Raw and processed sequencing data have been submitted to the GEO database (GSE88915). Further data analysis was performed using RStudio . Normalization of miRNA counts and differential expression analysis was performed using the R package DESeq2 (v1.8.2). The DESeq2 result tables are available in How to cite this article: Van Goethem, A. et al. Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples. Sci. Rep.6, 37876; doi: 10.1038/srep37876 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.