A report on the 13th International Bioinformatics Workshop held in Harbin, China, 5–6 August 2017. The International Bioinformatics Workshop (IBW), held every other year in China since 2003, has grown into an international forum for showcasing the most important breakthroughs in bioinformatics-related fields. At this year’s IBW, the topics of several presentations were at the center of attention, including novel functional features of genomes and transcriptomes, three-dimensional genome organization, and recent evolutions of mammalian and human genomes. Here, we summarize a subset of IBW’s presentations on these topics.Approximately 30% of the human genome comprises repetitive sequences known as transposable elements (TEs). Functional analyses of the human genome have paid little attention to TEs; however, the revelation that previously so-called “junk” DNA might be functional has aroused intrigue in this “dark matter” of the genome.Alu elements, Yi Xing  revealed one novel function of genomic dark matter. Alu-derived exons exhibit diverse alternative splicing patterns in various human tissues. As well as their role in the modulation of translation efficiency, some of these Alu-originated exons also encode new peptides. How do TEs shape human transcriptional networks? Ting Wang  gave the example of AluJb, a subfamily of Alu sequences, which, when inserted into the genome, gave rise to alternative promoters to activate oncogene LIN28B. Knocking out a specific copy of AluJb in a lung cancer cell line suppressed cell growth and migration.By showing the birth of new exons derived from the insertion of Wei Li  reported a novel mechanism of 3′ UTR shortening that leads to the repression of tumor suppressor genes by disrupting crosstalk with competing endogenous RNA. At least in part, this process is mediated by an RNA cleavage factor CFIm25, which may be responsible for regulating alternative polyadenylation sites on thousands of messenger RNAs.Synonymous single-nucleotide variants (sSNVs) are usually left out of analyses because of the absence of resulting amino acid changes. However, by using regSNPs-splicing software to prioritize sSNVs associated with RNA splicing, Yunlong Liu  found that disease-causing sSNVs are enriched in protein functional domains. Potential functional enrichment of intronic single-nucleotide variants was also discussed.https://bitbucket.org/liulab/trust/), a tool to assemble T-cell receptor hypervariable region sequences by assigning informative unmapped reads from tumor RNA-seq data into T-cell receptor genes. Wei Wang  proposed the Taiji pipeline (https://github.com/kaizhang/Taiji) to construct gene regulatory networks and identify key regulators of a specific cell stage by integrating multi-type high-throughput sequencing data including RNA-seq, open chromatin, and histone modifications.Yi Xing  presented rMATS-turbo, an updated version of rMATS that allows ultra-fast detection of differential alternative splicing and isoforms from replicated RNA-seq data. Shirley Liu  described TRUST  is endogenous non-coding RNA with covalently linked 3′ and 5′ ends that form a backsplicing structure. It is tissue-specific and evolutionarily conserved, suggesting a potential functional role. Fangqing Zhao  reported the prevalence of alternative splicing within circRNAs and their tissue-specific expression patterns. To help explore this line of work, the Zhao group developed a new method based on the backsplicing and reverse overlap features of circRNA. This method was able to recover ~ 80% full transcripts of circRNAs in cells (unpublished data). Li Yang  discussed the species-specific expression of circRNAs from an evolutionary perspective. He reported that the rapidly evolved SINEs (short interspersed nuclear repetitive DNA elements), especially RNA editing post-transcriptionally alters RNA sequences by substituting, deleting, or inserting nucleotides that can cause changes in RNA structure or the resulting protein product(s). Han Liang  reported the effect of A-to-I RNA editing on microRNAs (miRNAs) and its function in cancer. Liang and colleagues identified miR-200b through a pan-cancer analysis of TCGA transcriptomic data. The level of editing of this microRNA revealed distinct patterns associated with patient survival time compared with the primary miRNA. Specifically, unedited miR-200b inhibited epithelial–mesenchymal transition and suppressed tumor metastasis. In contrast, with only a single nucleotide modification in the mature region, the edited miR-200b was able to promote the migration and invasion of cancer cells by retargeting a new set of genes that included a key metastatic suppressor, LIFR. This striking example highlights the importance of RNA editing in cancer development.https://www.4dnucleome.org/) for revealing genome architecture and nuclear organization.Determining the interactions between different types of RNA, or between RNA and chromatin, is key to understanding their functions. Sheng Zhong  reported two techniques, MARIO and MARGI, for the massive detection of RNA–RNA and RNA–chromatin interactions in vivo. Zhong also introduced the plans of the 4D Nucleome consortium (http://yunliweb.its.unc.edu/HUGIn/), a unified web browser for visualizing and annotating Hi-C data from human primary tissues and cell lines. Zhihua Zhang  described Delta, a new 3D genome visualization tool, and DeDoc, a new method for calling topologically associated domains by merging and combining structural coding trees. Notably, DeDoc can stably detect topologically associated domains with just a few pieces of single cell Hi-C data.New methods and tools have been developed to analyze and present genomic interaction datasets. Yun Li  described HUGIn  presented PEP, a tool to predict enhancer–promoter interactions using only sequence-based features.Wenfeng Qian  discussed the relationship between genetic interactions (epistasis) and the order of eukaryotic genes on a chromosome. By extending theories from population genetics, Qian proposed the hypothesis that genetic interaction networks might drive the evolution of gene order. In support of his hypothesis, analysis of the global genetic interaction network recently published in budding yeast indeed revealed an anti-correlation between epistasis and gene distance. This was partially attributed to genes exhibiting positive epistasis having a tendency to translocate close to each other on a chromosome during evolution.EPAS1 and HBB that are associated with hypoxia tolerance, and suggested that this trait originated from Tibetan gray wolves.Two of the speakers described genome-wide association studies to investigate the genetic basis of high altitude adaptation in humans and the Tibetan mastiff dog from the Tibet Plateau. Shuhua Xu  investigated the genetic origin of high altitude adaption in Tibetans using deep-sequenced whole genome data. Using ArchaicSeeker, a tool developed by his own group, he suggested that the Tibetan people are an admixture of multiple populations, with an ancestry derived from both archaic and modern human groups. Xu also proposed a “fitness-borrow” hypothesis to explain the mechanism of altitude adaptation in Tibetans and Sherpas. Yixue Li  investigated the genetic basis of hypoxia adaptation in the Tibetan mastiff. He identified two loci for the genes https://github.com/genome/pindel), a tool for detecting complex indels and structural variations from next-generation sequencing data. Pindel-C detected complex indels in 285 cancer genes from The Cancer Genome Atlas, which were missed in previous studies. Interestingly, applying Pindel-C to whole genome sequencing data of 250 trio-families from the Genome of the Netherlands project, Ye also found that most germline mutations are of paternal origin.Ge Gao  reported on COPE, a genomic variant annotation tool that accounts for the cumulative effects of multiple variants within the same loci. COPE identifies multiple function-changing variants that are neglected by conventional tools from the 1000 Genomes dataset. Kai Ye  presented Pindel-C , bioinformatics emerged as an auxiliary tool in biomedical research and has grown into an independent discipline at the forefront of biological discovery and applications. IBW participants have become increasingly diverse over the years, perhaps reflecting the increasing collaboration between computational and experimental biologists as well as biomedical practitioners.


RNA dependent RNA polymerase (RdRp) is one of the most versatile enzymes of RNA viruses that is indispensable for replicating the genome as well as for carrying out transcription. The core structural features of RdRps are conserved, despite the divergence in their sequences. The structure of RdRp resembles that of a cupped right hand and consists of fingers, palm and thumb subdomains. The catalysis involves the participation of conserved aspartates and divalent metal ions. Complexes of RdRps with substrates, inhibitors and metal ions provide a comprehensive view of their functional mechanism and offer valuable insights regarding the development of antivirals. In this article, we provide an overview of the structural aspects of RdRps and their complexes from the Group III, IV and V viruses and their structure-based phylogeny. Their success results from the possibility of accommodating rapid changes via mutations, which aids in countering constant challenges imposed by the host physiology . Of the activity . The incactivity . In addiactivity .RdRps are multi-domain (α and β) proteins belonging to Structural Classification of Proteins (SCOP) class 2.7.7.48. They catalyze RNA-template dependent formation of phosphodiester bonds between ribonucleotides in the presence of divalent metal ions ,6,7,8.Many viral polymerases possess additional domains such as methyltransferase or endonuclease domain to carry out functions associated with RNA synthesis. The polymerase domain may also interact with other host factors for efficient polymerization and to discriminate activities such as genome replication and mRNA transcription ,9,10,11.In the current review, X-ray crystal structures of RdRps (available as of September 2017) belonging to PROSITE  accessioThe core RdRp domain consists of the thumb, palm and the fingers subdomains that are primarily involved in template binding, polymerization, nucleoside triphosphate (NTP) entry and associated functions A. The paPicornaviridae and Caliciviridae that use a primer to initiate replication have small thumb subdomains to facilitate the binding of primer such as viral protein genome-linked (VPg), while RdRps from Flaviviridae have large thumb subdomains to aid de novo initiation of replication [Flaviviridae, Leviviridae, Orthomyxoviridae, and Reoviridae with de novo initiation often possess a “priming loop” that emanates from the thumb subdomain and acts like a stabilizing platform during initiation [The thumb subdomain harbors residues that are involved in packing against the template RNA and stabilizing the initiating NTPs on the template . This sulication ,20,24,25itiation ,27,28,29itiation .Bovine viral diarrhea virus (BVDV) [Hepatitis C virus (HCV) [Pseudomonas phage φ6 (φ6) [Pseudomonas phage φ12 (φ12) [Birnaviridae [Picobirnaviridae [The fingers subdomain plays a significant role in setting the geometry of the active site. It serves to hold the template RNA in place and facilitates polymerization . The fins (BVDV) ,34, fourus (HCV) ,36,37 anus (HCV) ,39,40,41 φ6 (φ6) ,43 and P12 (φ12) . A twistaviridae ,45 and Paviridae . Surprisaviridae ,22,45,46A set of seven structural motifs, A to G, characterize the conserved structural component of RdRps indicating their pivotal role in catalysis ,48. The 2-4D in which the first aspartate is invariant in various RdRps. The second aspartate together with a conserved asparagine from motif B plays a crucial role in the discrimination of NTPs over deoxy NTPs by forming a hydrogen bond with 2′OH of the incoming NTP [Motif A, formed by a β-strand, continues either as a helix or a loop into the fingers subdomain . It housming NTP . A conseming NTP ,50,51. Tming NTP .Motif B of the palm subdomain assists the binding of the template RNA and in substrate discrimination . It is m2-4D align at the tip of the RRM aiding in efficient catalysis  [Motif E is a β-hairpin located at the junction of the palm and the thumb subdomain. It is also termed as “the primer grip” as it aids the correct positioning of the 3′ hydroxyl group of the primer for catalysis ,61. An a Qβ (Qβ) ,28.Motif F, formed by a loop and a β-strand, interacts with the phosphate group of incoming NTP. It comprises of positively charged residues that shield the negative charges of the phosphate groups of incoming NTPs by lying directly over the palm subdomain ,48. The Influenza A virus (FluA) [Influenza B virus (FluB) [Influenza C virus (FluC) [Motif G comprises a loop that is a part of the template entrance tunnel in ss (+) RNA viruses ,20 and fs (FluA) ,67 and Is (FluB) , Polymers (FluC)  and PA Cs (FluC) ,63.Reoviridae, this channel is formed by the residues of the N-terminal domain along with the fingers and thumb subdomains. It binds the 3′ end of (−) RNA during transcription and (+) RNA during genome replication [Flaviviridae such as JEV [Dengue virus (DENV) [Zika virus (ZIKV) [West nile virus (WNV) [Picornaviridae and Caliciviridae have a wider template channel and accommodate both a template and a protein primer [The RdRps have channels or tunnels that traverse in different directions connecting the catalytic centers with the exterior ,48. Theslication ,71. In Qlication . The Piclication . The temh as JEV , Dengue s (DENV) ,74,75,76s (ZIKV) ,78,79, Wus (WNV)  and HCV us (WNV) ,81,82. Tus (WNV) ,25,30. In primer ,20.Reoviridae, this channel is the largest of all, extending through the C-terminal domain [Reoviridae serves as the exit channel for the (+) RNA products of transcription [Cystoviridae [Birnaviridae [Picobirnaviridae [Arenaviridae family [Human picobirnavirus (hPBV) [Vesicular stomatitis virus (VSV) [Reoviridae [The RNA exit channel is formed by both the palm and the thumb subdomains and serves as the exit path for the template, as well as, newly synthesized RNA. In l domain . In the l domain . A uniqucription . Howeveroviridae ,44,83, Baviridae ,22, Picoaviridae  and the aviridae ,88,89,90aviridae ,50,51. Te family , Human ps (hPBV)  and Vesius (VSV)  is propooviridae .Bombyx mori Cytoplasmic polyhedrosis virus (BmCPV) [Although the core polymerase structure retains significant similarity in various groups, there are additional domains in individual viruses that support other processes accompanying RNA synthesis. λ3 polymerases of Reoviruses , Rotavir (BmCPV)  resemble (BmCPV) . The Bir (BmCPV) ,45 and P (BmCPV)  lack the (BmCPV) .Leviviridae is a tetrameric protein complex comprising of the RdRp (β subunit) and three host factors that include the translational elongation factors, EF-Tu and EF-Ts and ribosomal protein S1  [Bunyaviridae share distinctive resemblances to the heterotrimeric RdRps of Influenza viruses [In the segmented ss (−) RNA viruses from x (vRNP) ,50,51. T viruses ,63. Thei viruses ,63. The  viruses  that pos viruses . The stu viruses . The cor viruses ,98. The  viruses ,99.http://proteinmodel.org/AS2TS/STRALCP/stralcp.cgi) for clustering protein domains based on their structural similarities  [Birnaviridae and Permutotetraviridae permute cyclically due to the non-canonical C-A-B arrangement of the motifs instead of the regular A-B-C leading to an altered active site geometry [Birnaviridae that is distinct from other dsRNA viruses as apparent in the phylogenetic profile   also shageometry ,45,106.  profile C.Picobirnaviridae has the characteristic GDD motif and the canonical arrangement of A-B-C motifs [Unlike Birnaviral RdRps, the RdRp of hPBV from C motifs . It, on C motifs  in posseC motifs ,30. The C motifs  is possiLeviviridae group [Cystoviridae [Reoviridae [Reoviridae have evolved to possess a four-channeled architecture unlike the three-channeled ones for the RdRps employing semi-conservative RNA synthesis [Cystoviridae, Flaviviridae, Picobirnaviridae, and Birnaviridae share the same sub-clade, which is distinct from Reoviridae in the structural dendrogram  strand of RNA helps in distinguishing it from the (−) strand and engaging the RdRp close to the 3′ end of the (−) strand to facilitate transcription [Detailed studies with the λ3 polymerase of MRV3 in complex with the cap analog, (7-methyl-guanosine-5’-triphosphate-5′ guanosine), RNA oligonucleotides, rNTPs and 3’-deoxy-NTP provide valuable insights into the mode of initiation, elongation, and encapsidation of dsRNA . The stucription ,109. A ucription . The strcription .X-ray crystal structures of the RdRps of Qβ and φ6 in complex with template RNA, NTPs, and their analogs are available. The structures suggest the molecular basis for de novo initiation, elongation and consequences of calcium binding ,95. The 2+ of elongation complexes of φ6 RdRp [In bacteriophage Qβ, the crystal structure of the core replicase in complex with a template RNA ending with CCA-3′ and complementary 7- or 8-mer RNA oligonucleotides are available . In many φ6 RdRp .Many RNA/NTP bound structures are available for the Group IV viruses. In Calicicviruses, the structures of RdRp complexes of hNoV reveal exciting approaches for inhibitor design using nucleotide analogs ,113,114.pol with GTP (PDB ID: 1ra7) provided deeper insights into the mechanism of proteolysis-dependent activation of the polymerase [Picornaviridae reveal new regions close to the template binding site at the interface of thumb and fingers that are suitable for the development of antivirals [Foot-and-mouth disease virus (FMDV) [A thorough study of the mode of NTP binding, elongation and catalysis has come from the structures of picornaviral RdRp complexes . The strlymerase ,116. Tholymerase ,117. Furlymerase ,117. Simlymerase ,23,116. tivirals . Structus (FMDV) ,118, PV s (FMDV) ,115,116,s (FMDV) ,115, Hums (FMDV) ,115, Ents (FMDV)  and Entes (FMDV) ,117 haves (FMDV) . These ss (FMDV) . Additios (FMDV) .Due to the severe health concerns associated with both HCV and DENV, the structural studies on RdRp complexes of Flaviviruses, particularly HCV, have surpassed those from any other group or individual virus. Pegylated interferons and ribavirin were routinely used for the treatment of HCV infection before 2013. However, the treatment is now replaced by Direct Acting Antivirals (DAAs) . Among tAn exhaustive study involving complexes of RdRp of HCV with RNA templates, primers, NTPs, nucleoside analogs and catalytic metal ions have provided useful insights into the role of the priming loop of the thumb subdomain and the C-terminal membrane-anchoring linker during de novo initiation and elongation . TogetheThe NNIs of HCV and DENV affect polymerase activity by interfering with crucial conformational changes associated with catalysis and elongation by preventing the contacts between the thumb and the fingers subdomain ,122. Fiv50 of ≈700 µM. In a cell-based assay, another lead candidate that has a replacement of the carboxylic acid moiety with an isosteric acylsulfonamide displayed antiviral activity at low micromolar concentrations against all four DENV serotypes [Five PDBs represent structures of complexes of NNIs from the Novartis’ fragment collection of DENV RdRp obtained by X-ray crystallography . The inherotypes . Many of2-4D and GDD) and the seven other structural motifs (A–G) are found to be conserved. The core structural elements of RdRps include those of DNA and RNA polymerases formed by the fingers, palm and thumb subdomains. Presence of additional domains and association with host proteins aid the RdRps in various processes associated with RNA synthesis. The structural comparisons of viruses from different groups indicate how the conformational elements have evolved to meet the requirements arising out of a change in genome or dependence on a primer. Analysis of ligand complexes provides deeper insights into the role of metal ions and the function of structural elements in template recognition and binding. The study of inhibitor complexes helps to take stock of the structural data of potential drug candidates that are currently available and understand their modes of inhibition.The structures of monomeric RdRps from different viruses that are available in the PDB provide useful insights regarding the conservation of the core structural elements required for the functioning of this versatile molecule. Although the sequences have diverged significantly, the catalytic elements including the sequence motifs (DX


Approximately 9% of cancer-related deaths are caused by colorectal cancer. Liver metastasis is a major factor for the high colorectal cancer mortality rate. However, the molecular mechanism underlying colorectal cancer liver metastasis remains unclear. Using a global and multidimensional integration approach, we studied sequencing data, protein-protein interactions, and regulation of transcription factor and non-coding RNAs in primary tumor samples and liver metastasis samples to unveil the potential bridging molecules and the regulators that functionally link different stages of colorectal cancer liver metastasis. Primary tumor samples and liver metastasis samples had modules with significant overlap and crosstalk from which we identified several bridging genes (e.g. KNG1 and COX5B), transcription factors (e.g. E2F4 and CDX2), microRNAs (e.g. miR-590-3p and miR-203) and lncRNAs (e.g. lincIRX5 and lincFOXF1) that may play an important role in the process of colorectal cancer liver metastasis. This study enhances our understanding of the genetic alterations and transcriptional regulation that drive the metastatic process, but also provides the methodology to guide the studies on other metastatic cancers. Colorectal cancer (CRC) is the second most common cancer in men, the third most common cancer in women, and accounts for approximately 9% of all cancer deaths. Liver mTumor invasion and metastasis are dynamic processes involving multiple steps. These processes primarily include: 1) massive proliferation of tumor cells in primary lesions, 2) the acquirement of metastatic genes by a small population of tumor cells, 3) the detachment of tumor cells from primary lesions, 4) the invasion of the basement membrane, 5) entry into and exit from the circulatory system, and 6) the colonization and formation of secondary tumors at a distant site [Over decades of intensive research, several individual functional molecules have been identified in the process of CRC liver metastasis, such as KRAS, BRAF, and EGFR, 6. HoweIn this study, we used a global and multidimensional integration strategy to analyze the sequencing data, protein-protein interaction (PPI) and the regulation data of transcription factor (TF) and non-coding RNA from samples obtained from primary non-metastatic colorectal tumor (PNMCT), primary metastatic colorectal tumor (PMCT), and their paired metastatic CRC samples in the liver (LMCT). This approach enabled us to identify the potential bridging molecules that play a role in the process of CRC liver metastasis including modules with significant overlap and crosstalk. Furthermore, some potential molecular targets including TFs, miRNAs, and lncRNAs were identified through the establishment of a regulatory network of modules. Our results from this comprehensive analysis demonstrate a new methodology for studying the molecular mechanisms of CRC liver metastasis.This study was approved by GEN (Genetics/Population Research/Investigator Initiated Studies) Search Ethics of McGill University Health Center. . The patient consent was waived by the ethics committee as our study only used archived tissue and had no to patient care. With the approval of the institutional ethics review board, we collected 10 PNMCT samples, 9 PMCT samples and 9 LMCT samples from 19 patients with CRC who received the diagnosis and treatment in McGill University Health Center. This investigation was conducted according to the Declaration of Helsinki. Affymetrix Human Transcriptome Array 2.0 was used to sequence the gene expression of these samples. The sequencing data was uploaded to the GEO database (No. GSE72718).2Fold Change|>|log21.2| and p value<0.05. Moreover, we established the heatmap of DEGs. Finally, we used the principal component analysis (PCA) method to confirm the correlation between disease and DEGs[Based on these sequencing data, we used the R limma package to identify the DEGs in PMCT (PMCT vs PNMC) and LMCT (LMCT vs PNMCT). The threshold of DEGs is |log and DEGs.http://string-db.org/) and Cytoscape software were used to establish the PPI networks of PMCT and LMCT[The STRING database V10  as: (i) at least two regulations between the regulator and each module of the pair and (ii) significant enrichment of targets for each regulator per module with a p value cutoff of 0.05 . In thishttps://cancergenome.nih.gov) to analysis the expression of target genes and their effect on the survival rate of CRC patients. Cbioportal  was used to analysis 276 CRC samples in TCGA database. Enter the information to the web page of Cbioportal as following: 1) Select Genomic Profiles: mRNA Expression data; 2) Select Patient/Case Set: Tumors with sequencing and CAN data (212); 3) Enter Gene Set: the target genes; 4) Click on “OncoPrint” and “Survival”. Logrank test was used to analysis the significance of survival rate. Finally, we used Illustrator software to combine the survival rate files.To verify the results of the overlap and crosstalk networks, we used TCGA database  to perform a Gene Ontology (GO) analysis on the gene nodes in the PPI network. The results show that GO terms with a p value cutoff of 0.01 were primarily involved in the “positive regulation of cell proliferation” and “mitochondrial transport”  but also demonstrated crosstalk with LMCT-7 . StudiesTo clarify the underlying transcription and post-transcription regulation networks of the modules with overlap and crosstalk, we explored the miRNAs, lncRNAs, and TFs that are responsible for the regulation of genes in these 2 modules. A total of 4 upstream miRNAs, 9 upstream lncRNAs, and 69 TFs that met the conditions were predicted among genes in the 82 overlapping network modules . DEGs inWe found that some of the TFs that we identified have already been implicated in the regulation of liver metastasis in the setting of CRC. For example, CDX2 is a TF with tumor suppressor functions that has been shown to be minimally expressed in CRC cell lines; the over-expression of CDX2 significantly inhibits the invasive ability of CRC cells. PancionIn addition to TFs, non-coding genes are also very important for the post-transcriptional regulation of overlapping and crosstalk networks. In the transcription regulation networks of overlapping modules, all of the miRNAs that we identified are closely associated with tumor metastasis. For example, miR-216b and miR-486-5p inhibit invasion and metastasis of liver cancer cells, 46, whiIn contrast to miRNAs that have been extensively investigated, studies on the functions of lncRNAs are still at the initial stage, and the functions of many lncRNAs are still unclear. However, recent studies have indicated that some of our screened lncRNAs possess regulatory functions in the metastasis of CRC. For example, MALATA1 and BANCR can promote the migration and invasion of CRC, 57, whiWe used TCGA database to analysis the expression of target genes in overlap and crosstalk network and their effect on the survival rate of CRC patients. The results showed that five target genes in the overlap and crosstalk networks were differentially expressed in CRC samples and could effect the survival rate of CRC patients.  Among 276 CRC samples in TCGA database, we found the CRC patients with alteration in KNG1 (p = 0.0215) or MYO5C (p = 0.0312) had lower survival rates than the CRC patients with normal KNG1 or MYO5C expression. Moreover, the CRC patients with alteration in JAG1 (p = 0.0077) or ACTN2 (p = 0.0233) or MYCN (p = 0.0297) had lower survival rates than the CRC patients with normal JAG1 or ACTN2 or MYCN expression.  In this High-throughput next generation sequencing offers a powerful tool for studying the underlying genetic alterations of a disease process. Using this technology, we studied the genetic changes that underlie different stages (from PNMCT through PMCT to LMCT) of CRC liver metastasis. To fully unveil the global perspective provided by high-throughput sequencing data, we applied an integration methodology based on sequencing data, PPI networks, transcription regulation, and some non-coding RNA prediction databases to discover bridging targets of CRC from a non-metastatic state to a metastatic state.Our established overlapping networks revealed 7 overlapping genes between the related modules of PMCT and LMCT. These genes might regulate a variety of biological processes associated with tumor metastasis including the regulation of GPCR signaling, cell proliferation, and energy metabolism. As demonstrated, some overlapping genes such as KISS1R, COPS5, and PTEN have been shown to have important regulatory functions in the process of CRC metastasis , 26, 27.Based on a comprehensive strategy integrating many types of databases and statistical algorithms, we constructed transcription regulation networks including TFs, miRNAs, and lncRNAs between the PMCT and LMCT groups, each of which had modules with considerable overlap and crosstalk. After these 2 networks were combined, we found that 9 of the top 10 TFs could regulate gene nodes in these 2 networks. Some of these genes, such as CDX2, INI1, and E2F1, have been shown to influence the metastasis of CRC–41, whicIn summary, using next generation sequencing data from tumor samples, we used an integrated approach to explore the potential molecular targets that play a bridging role in the process of CRC liver metastasis. Our results demonstrate the power of a global and multidimensional systematic approach on the discovery of potential molecular mechanisms of tumor metastasis. This strategy not only unveils the dynamic links between PNMCT, PMCT and LMCT, but could also be applicable to the study of other tumor metastasis, such as breast cancer with bone metastasis, hepatocellular carcinoma with lung metastasis and lung cancer with brain metastasis. In addition to further study on validated molecules, more efforts should be made to study the remaining potential molecular targets . These new molecular targets may also play an important role in CRC liver metastasis and could be new tools for research on mechanisms in the future.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


More and more ribonucleases were discovered in recent years with the progress of technology. In the present study, we found that the uncharacterized C19orf43, a novel interacting protein of human telomerase RNA (hTR), digested T7 transcribed RNA, total cellular RNA and RNA oligos but not DNA. Thus we named this new RNase as hTRIR (human telomerase RNA interacting RNase). Genetic analysis showed that hTRIR is conserved among eukaryotic species and widely expressed in different cell lines. The RNase activity of hTRIR works in a broad temperature and pH range while divalent cations are not required. The conserved C-terminus of C19orf43 is necessary for its activity. Finally, we found that hTRIR cleaves all four unpaired RNA nucleotides from 5′ end or 3′ end with higher efficiency for purine bases, which suggested that hTRIR is an exoribonuclease. Taken together, our study showed the first evidence of the novel function of hTRIR  The metabolism of RNA transcripts requires a large number of distinct ribonucleases which includes endoribonucleases and exoribonucleases124678111215http://www.genecards.org/cgi-bin/carddisp.pl?gene=C19orf43). C19orf43 is highly expressed in nasal epithelium, peripheral blood mononuclear cells, breast, and T lymphocyte. An acetylation site was detected at lysine-146 in human C19orf43 protein with large scale analysis in cellular proteomics by mass spectrum, but the function of this modification is unknownThe uncharacterized human protein C19orf43 is located at Chromosome 19: p12 and encodes a protein of 176 amino acids with a molecular weight of 18,419 Dalton17UbC is a stress-inducible polyubiquitin gene and maintains cellular ubiquitin (Ub) levels during stressSNRPA, RPL36, UbC and SF3A2 have been reported to interact with C19orf4324Human telomerase RNA (hTR) is one of the subunits of telomerase holoenzyme28303133To identify the 3′ end processing proteins of hTR, HeLa-S3 cell nuclear extract (NE) was prepared and partially purified by heparin, cation exchange and gel filtration chromatography. The uncharacterized C19orf43 protein was found in the crude purified complex by mass spectrum. Genetic analysis showed that C19orf43 is conservative among eukaryotic species. Incubation of C19orf43 with T7 transcribed RNA resulted in the degradation of RNA, which suggested that C19orf43 possessed ribonuclease activity. Thus we named this protein as hTRIR (human telomerase RNA interacting RNase). Further study showed that hTRIR not only cleaved T7 transcribed RNA but also total cellular RNA. The RNase activity of hTRIR worked in a broad pH range from 6.5 to 12 and divalent cation was not required. The C-terminus contains the key domain for RNase digestion activity. Importantly, hTRIR cleaves all four RNA nucleotides from 5′ or 3′ end with preference for A/G, suggesting that hTRIR is a novel exoribonuclease.Proper 3′ end processing of a RNA nascent transcript by endoribonuclease or exoribonuclease is critical for the localization, function and stability of the mature RNA35http://www.proteinatlas.org/ENSG00000123144-C19orf43/tissue). To further confirm the expression of hTRIR protein, polyclonal antibody against hTRIR was developed by immunization of rabbit with purified protein. Several cell lines were cultured and total protein was harvested to detect hTRIR expression by using western blot  (We next did text mining by STRINGdb.org/)  to searcin vitro and incubated with 0.1 μg or 1 μg purified hTRIR at 37 °C for 30 min in the digestion buffer . RNA was separated by 7 M urea 6% polyacrylamide gel electrophoresis (PAGE) and the signal was detected by Typhoon cyclone. To our surprise, 53% of T7 transcribed hTR was degraded when 0.1 μg hTRIR was incubated with hTR and over 90% of the transcripts were digested by 1 μg hTRIR in the absence of ATP  tail. Whether hTRIR digests cellular RNA is unknown. To this end, 2 μg total RNA from HeLa-S3 cells were incubated with 0.1, 0.2, 1 and 2 μg hTRIR. Over 99% 28 S and 18 S RNAs were degraded when the amount of hTRIR increased to 2 μg . No RNA cleavage was observed when RNA was incubated with those proteins, which indicated that it was the function of hTRIR resulting in the degradation of RNA.The digestion condition was optimized by different time, pH and ions since hTRIR worked in the digestion buffer which contains 25 mM NaDifferent RNases cleave RNA with different mechanism42548in vitro. However, whether hTRIR played a role in the biogenesis of hTR in vivo is still unknown. Further study showed that hTRIR was able to cleave cellular total RNA and RNA oliogs in vitro. Whether hTRIR is involved in other cellular process is unclear yet. Our study does not reveal the key amino acid for hTRIR, which is still under investigation.RNase A super-family members not only cleaves RNA, but also suppresses cancer and tumor101151in vitro. How hTRIR affects the cell and whether it participates in other cellular process need to be elucidated in the future. Our study provided the clue to look into the function of hTRIR in vivo.In conclusion, hTRIR protein is conservative among eukaryotic species and possesses the function of exoribonuclease This study was performed in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals according to the regulation in the People’s Republic of China. Based on “Guideline for Animal Care and Use, Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS)”, The animal used in the experiment was bred and maintained under specific pathogen-free conditions at the Central Animal Laboratory of Wuhan Institute of Virology, Chinese Academy of Sciences . All animal experiments were approved by the Institutional Animal Ethical Committee of WIV, CAS . All experimental protocols were approved by the Ethical Committee of WIV, CAS.2. BxPC-3  pancreatic cancer cells were cultured in Gibco RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C, 5% CO2. HeLa-S3 cells were suspension cultured in Minimum Essential Medium (MEM) Eagle Joklik, w/L-Gln w/o Ca2+ (Life science), containing 10% fetal bovine serum at 37 °C, 5% CO2.HeLa-S3 , HEK293T , VA13 , Huh 7, Hep G2 , M2 , MEFs , BHK-21 , WRD (kind gift from Dr. Yunning Sun), and MDCK  cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium  containing 10% fetal bovine serum at 37 °C, 5% CO5 cells/ml by low speed centrifugation . Cells were resuspended and broken with dounce homogenizer (B pestle). Nuclei were spun down at 25,000 g, 4 °C, for 20 min. The nuclei pellet was extracted with KCl to a final concentration of 0.24 M. The nuclear extract was ultracentrifuged at 40000 rpm (Beckman), 4 °C for 30 min. The supernatant was dialyzed twice against Buffer D  for 4 hours. The extract was snap-frozen in liquid N2 and stored at −80 °C.HeLa-S3 nuclear extract was prepared as previously describedTotal RNA from HeLa-S3 was isolated with Trizol reagent (Ambion) according to the manufacturer’s instruction.hTRIR cDNA was synthesized with SuperScript™ III Reverse Transcriptase (Invitrogen) according to the manufacturer’s handbook.hTRIR  open reading frame, nt 1–528 was amplified and inserted into pET-33b(+) prokaryotic expression vector. hTRIRA, hTRIRB, hTRIRC, and hTRIRD were constructed by insertion of nt 163–528, nt 163–444, nt 1–330, nt 331–528, into pET-33b(+) vector, respectively. All the primers in plasmid construction were listed as follows: hTRIR , hTRIRA , hTRIRB , hTRIRC , hTRIRD Protein expression and purification were performed as previously describedPurified hTRIR protein was applied to immunize rabbit according to previously describedCells were seeded into 60 mm culture plate. After 48 hours, cells were harvested when cell confluence reached to 90%. Cell lysate were separated with 15% SDS-PAGE, transferred to nitrocellulose and probed with hTRIR polyclonal antibody. Luminescent signals were detected with SYSTEM GelDoc XR + IMAGELAB (Bio-Rad).32P-GTP (PerkinElmer) labeled RNA was transcribed with T7 polymerase (Thermo scientific) according to manufacturer’s instruction.α-T7 transcribed RNA, total cellular RNA or RNA oligos were incubated with different amount of purified hTRIR in digestion buffer  glycerol) at 37 °C for 30 min or indicated time. RNAs were harvested and separated by 7 M urea polyacrylamide gel electrophoresis (PAGE) or 1.5% agarose (Biowest) gel. T7 Transcribed RNA signals were detected by Typhoon cyclone (PerkinElmer). Total cellular RNA signals were detected by SYSTEM GelDoc XR+ IMAGELAB (Bio-Rad). Small RNA was stained with stain-all (Sigma) and scanned with Epson scanner.Total amount of 1 μg different types of DNA were incubated with 0.1 to 5 μg of purified hTRIR protein at 37 °C for 30 min. DNA was separated with 1% agarose (Biowest) gel and detected with SYSTEM GelDoc XR + IMAGELAB (Bio-Rad).t test was applied to determine differences in DNA and RNA digestion assay. p < 0.05 was significant and marked with *.Experiments were repeated at least three times. Quantification data were presented by mean and stand deviation. Student’s How to cite this article: Xie, J. et al. Identification of an RNase that preferentially cleaves A/G nucleotides. Sci. Rep.7, 45207; doi: 10.1038/srep45207 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER.DOI:http://dx.doi.org/10.7554/eLife.22750.001 RNA silencing is a conserved posttranscriptional gene regulatory mechanism in eukaryotes. At the core of RNA silencing are small RNAs, microRNAs (miRNAs) or small interfering RNAs (siRNAs), and their effector ARGONAUTE (AGO) proteins. Many observations document the membrane association of plant and animal AGO proteins , but theC. elegans is signal amplification, in which primary siRNAs from transgenes or viruses guide the production of secondary siRNAs from target RNAs through the activities of RNA-dependent RNA polymerases (RdRP) (A well-documented feature of RNA silencing in plants and s (RdRP) . This reArabidopsis, eight noncoding TAS loci generate phasiRNAs in this manner and, as the phasiRNAs regulate target genes in trans, they are termed trans-acting siRNAs (ta-siRNAs)  and pentatricopeptide repeat (PPR) genes are targeted by 22-nt miRNAs and produce phasiRNAs  from target transcripts (reviewed in ). Upon m-siRNAs) . Most mi-siRNAs) ; the TAS trigger . In a ‘t trigger . The thrr miR390 , a miRNAr miR390 . In addihasiRNAs .TAS3 RNA is bound by ribosomes, and ribosomes on TAS3 RNA are stalled by AGO7 at the miR390-binding site, implicating that ta-siRNA biogenesis from TAS3 occurs on polysomes . We found an intriguing difference between miRNAs and siRNAs in their membrane-cytosol partitioning. We showed that all miRNAs, including 22-nt miRNAs, were associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The plant miRNA effector AGO1 associated with membranes in part in an RNA-independent manner, recruited miRNAs to membranes, and exerted its endonuclease activity on MBPs. Reduced membrane-association of 22-nt miRNAs in an We previously uncovered an integral ER membrane protein (named AMP1) as required for miRNA-mediated translational repression of target RNAs and showed that a few examined miRNAs and their target transcripts were present on MBPs . To gainAs the M fraction was a crude membrane preparation, and our previous study implicated the rough ER as the site of miRNA-mediated translational repression , we sougnrpd1-1 mutant (NRPD1 encodes the largest subunit of Pol IV (nrpd1-1 lacks most 24-nt P4siRNAs (nrpd1-1 (reads per million rRNA fragments).Because the M and T samples differed drastically in the composition of small RNAs, normalization against total reads were not appropriate if comparisons of sRNA levels between the two sample types were to be made. To establish a reasonable normalization method for such comparisons, we utilized published sRNA-seq libraries from wild type and the 1 mutant . NRPD1 ef Pol IV ; the sRNP4siRNAs   also had higher levels in MBP  between MBP and TP samples. This analysis revealed higher numbers of hyper (MBP>TP) than hypo (MBP<TP) differential sRNA regions (DSRs) . At the AS genes .MIR genes representing 24 miR families   . Of thesld type) . By examld type) . 22-nt mld type) .10.7554/dcl1 mutant but not in a dcl2 mutant  . Note thAGO1 partially co-localizes with ER and co-fractionates with microsomes and MBPs . We examDCL1 and HYL1 (CSD2 RNA (a target of miR398) was eliminated by RNase I treatment  and two for the microsomal fraction (M) in this mutant  in ago1-27 , miR472 and miR825* targeting NBS-LRR genes, and miR828 targeting TAS4. Among these miRNAs, miR173, miR393 and miR828 are predominantly 22 nt long while the others are predominantly 21 nt long with 22-nt isoforms. We also identified two new phasiRNA-generating loci: CIL2 (At3g23690) encoding a basic helix-loop-helix protein and being targeted by miR393, and At5g43740 encoding an NBS-LRR protein and being targeted by miR472  were nearly eliminated in ago1-36  was not reduced (they were in fact increased) in ago1-27  with MBPs. In brief, MBPs were first isolated  and thenTAS transcripts, which were thought to be non-protein-coding, were associated with MBPs. Although the entire TAS transcripts were associated with MBPs, as revealed by RNA-seq of MBP RNAs  wild-type (Columbia accession), ago1-27 , 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol, and a complete proteinase inhibitor cocktail (Roche)), and the cell lysate was filtered by 2 layers of miracloth and centrifuged at 10,000 g twice to remove debris. A 100 µl aliquot was taken from the clarified supernatant as input, and the rest was applied onto a sucrose step gradient (2.5 ml 20%/2.5 ml 60% sucrose), and centrifuged at 100,000 g in an SW41 rotor (Beckman coulter) for 1 hr. Microsomes were recovered from the interface of the two sucrose layers, diluted with 10 volumes of MEB and precipitated by centrifugation at 100,000 g for 30 min.The isolation of microsomes and MBPs for small RNA sequencing was performed as described . In brie2, 1.75M sucrose, 5 mM DTT, 50 μg/ml cycloheximide, and 50 μg/ml chloramphenicol) and centrifuged at 170,000 g for 3 hr. The pellet was dissolved with resuspension buffer  and the solution was transferred into a new tube as the MBP fraction. An aliquot of the MBP was subjected to 15–50% sucrose gradient centrifugation for polysome integrity evaluation, and the rest was used for sRNA and mRNA sequencing.To isolate MBP, the microsome preparation was lysed with 8 ml polysome isolation buffer . The lysate was kept on ice for 30 min, and then loaded on the top of a sucrose cushion 2, 50 mM EGTA, 0.25M sucrose, 10 mM DTT, 50 ug/ml cycloheximide, 50 ug/ml chloramphenicol and 2.5 U/ml superaseIN). The slurry was filtered by 2 layers of miracloth and centrifuged twice at 10,000 g for 10 min to remove debris. Microsomes were pelleted by centrifugation at 30,000 g for 30 min in a Beckman SW28 rotor. The supernatant was transferred into a new tube as the cytosol fraction, and the microsome pellet was dissolved with 8 ml extraction buffer supplemented with 1% v/v Triton X-100. Both the cytosol and microsomal fractions were centrifuged again at 30,000 g for 30 min to remove any residual membranes, and the supernatant was loaded onto an 8 ml 1.75M sucrose cushion as described in the 'Microsome and MBP isolation' section and centrifuged at 170,000 g for 3 hr. The FP and MBP pellets were resuspended with 400 µl resuspension buffer as described in the 'Microsome and MBP isolation' section, and polysome integrity evaluation was conducted as before.A detailed protocol for the isolation of cytosol, microsome, free polysomes (FP) and membrane-bound polysomes (MBP) may be found at Bio-protocol . The simNote that this procedure does not recover MBPs that are as pure as the procedure described above, but allows the simultaneous recovery of both cytosol and microsomes. This MBP isolation approach was only used when FP and MBP transcriptomes were compared. RNA was extracted from the cytosol and microsomal fractions by TRI-reagent (Molecular Research Center) and subjected to RNA-seq (see below).Total cellular polysomes were isolated as previously described . Briefly1.5 g seedlings were ground in liquid nitrogen and the powder was resuspended in 4 ml MEB buffer. The slurry was clarified by passage through two layers of miracloth and centrifugation at 10,000 g for 10 min twice at 4°C. The supernatant was subjected to further centrifugation at 100,000 g for 1 hr at 4°C to separate the cytosol (the soluble) and microsomal (the pellet) fractions. The pellet was washed by the same amount of MEB once again to avoid cytosol contamination of the microsomal fraction.AB_2224930; AGO4: AgriSera Cat# AS09 617 RRID:AB_10507623). To test the RNA dependence for AGO1’s membrane association, the clarified plant extract was treated with either RNase inhibitor (as a control) or 80 units/ml RNase I at room temperature for 1 hr, after which microsomes were isolated and subjected to RNA and protein analyses as described above.AGO1 and AGO4 were detected by western blotting with anti-AGO1 or anti-AGO4 antibodies (AGO1: AgriSera Cat# AS09 527 RRID:AB_2060757).To detect AGO1’s association with MBPs, MBPs were isolated as described in the ‘Microsome and MBP isolation’ section, and subjected to sucrose gradient centrifugation to separate the light fraction containing the 40S, 60S, and 80S ribosomes from the polysomes. Proteins were isolated from these fractions using TRI reagent (Molecular Research Center) following manufacturer's instructions. To determine the RNA-dependence of AGO1’s MBP association, 1000 O.D.260 units of MBPs were treated with 1500 units of RNase I followed by sucrose gradient centrifugation. The light fraction containing the 40S, 60S, and 80S ribosomes and the heavy fraction containing polysomes were recovered. Proteins were isolated from these fractions with TRI reagent following manufacturer's instructions and subjected to western blotting to detect AGO1 and the ribosomal protein L13 with commercial antibodies (AgriSera Cat# AS09 478 RRID:AB_2336883) for 2 hr at 4°C with gentle rotation, and washed by IP buffer four times. The beads were boiled with 1x protein loading buffer, and the proteins were subjected to western blotting to detect YFP-SEC12, HSC70, ARF1, SYP22, and PEPC using commercial antibodies . Using the same procedure, ER IP was performed followed by RNA isolation and sRNA sequencing .1 g seedlings expressing YFP-SEC12 were ground in liquid nitrogen into fine powder, and the powder was resuspended with 2 ml IP buffer containing 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.3M sucrose, 10% glycerol, and 1x proteinase inhibitor (Roche). 100 µl supernatant was saved as the input after clarification by 10,000 g centrifugation at 4°C, and the rest was used for immunoprecipitation. Briefly, the extract was pre-cleared by Dynabeads (Invitrogen), incubated with anti-GFP antibodies  to detect YFP-SEC12.Microsomes from YFP-SEC12 transgenic plants were isolated, resuspended with MEB buffer, and digested with 10 ng/µl proteinase K (Fermentas) in the presence or absence of 1% Triton X-100 for 20 min on ice. The reactions were stopped by the addition of phenylmethylsulfonyl fluoride to a final concentration of 5 mM and subjected to western blotting using anti-GFP antibodies  was used. To IP AGO1 from microsomes, microsomes were first isolated and dissolved in IP buffer containing 1% of Triton X-100. The microsome lysate was then diluted by 10 volumes of non-detergent-containing IP buffer to reduce the Triton X-100 concentration to 0.1%, and immunoprecipitation was performed. After washes, the beads were resuspended in the reaction buffer for slicer activity assay (see below).To IP AGO1 from total cell extracts, the same procedure as that of YFP-SEC12 IP was applied except that AGO1 antibody ). The reaction mix was incubated at room temperature for 2 hr. RNA was then extracted with TRI-reagent and resolved in a 5% polyacrylamide/7M urea gel, and 32P signals were detected with a Phosphoimager.For the slicer activity assay, part of the nd PHB-R . In vitrTo detect the 3' cleavage products from miRNA targets, 5' RACE was performed using the GeneRacer kit (Invitrogen). Total RNAs were directly ligated with the GeneRacer RNA oligonucleotide and reverse transcription reactions were carried out with an oligo dT primer. Seminal quantitative PCR was performed with gene specific primers  to quant30 µg of RNA was resolved on 15% urea-PAGE gel, and the 18–30-nt region was excised from the gel. Small RNAs were recovered by soaking the smashed gel in 0.3M NaCl overnight, followed by precipitation with ethanol. Small RNA libraries were constructed following instructions from the Illumina Truseq small RNA library preparation kit (Illumina). The libraries were sequenced on an Illumina Hiseq 2500 at the UC Riverside Genomics core facility.RNA-seq libraries were constructed using the NEBNext mRNA Library Prep kit (NEB). Briefly, polyA+ RNAs were isolated with the Dynabeads mRNA DIRECT Purification Kit (ThermoFisher Scientific), and fragmented to approximately 200 nt. cDNA was synthesized with random primers, and DNA adaptors were ligated to both ends of the double-stranded cDNAs. PCR was performed with primers complementary to the sequences in the DNA adaptors to generate RNA-seq libraries, which were sequenced on Illumina Hiseq2500.http://www.arabidopsis.org) using ShortStack v3.4 (reads per million of 45S rRNA reads) for each window, and comparison was conducted by the R package 'DESeq2' (Reads from Illumina sRNA-seq were first processed to remove the 3' adaptor sequences (TGGAATTCT or AGATCGGAA) and then size-selected (18 to 42 nt) using cutadapt v1.9.1 . Adaptorack v3.4  with par'DESeq2' .TAS gene regions  were counted and summed separately for each size class . Windows that harbor P4siRNAs were determined as described  in 400 µl digestion buffer  with gentle shaking at room temperature for 1.5 hr, and the digestion product was centrifuged in a 15–50% sucrose density gradient at 300,000 g for 1.5 hr. A gradient fractionation system (ISCO) was used to collect the monosome fraction. Monosomal RNAs were extracted with TRI Reagent (MRC), and resolved on 15% Urea-PAGE. RNAs corresponding to 25–35 nt were recovered from the gel and phosphorylated by T4 polynucleotide kinase (NEB). Next, the monosomal RNAs were further polyadenylated with PolyA Polymerase (NEB), and reverse transcribed with the primer oligodT linkerM , followeRibo-seq and MBP mRNA-seq datasets were processed using Tophat 2.0 with default parameters . Files cThe genomics datasets were deposited at NCBI GEO under the accession number GSE82041. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Gary Ruvkun (Reviewer #3).Thank you for submitting your article "Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this paper, you combined genomic approaches with cellular fractionation to investigate the global patterns of cytoplasmic partitioning of small RNAs and "long" RNAs. You show that miRNAs and their target RNAs are associated with membrane-bound polysomes (MBPs). Moreover, cleavage activity and products are detected in microsomes and MBPs. You propose that miRNA cleavage occurs on MBPs. You further show that phasiRNA-triggering 22-nt miRNAs and their targets including non-coding TAS transcripts are also present on MBPs and propose phasiRNA biogenesis is initiated on MBPs.The reviewers agreed that such findings should in principle be published at the highest levels. While the reviewers furthermore agreed that the data presented are clear and thorough, they also agreed that a couple of additional experiments would be essential to improve the manuscript.Essential revisions:1) Use dcl1 mutants to test whether miRNAs are required for the membrane association of AGO1. Alternatively, examine whether small RNA binding-deficient AGO1 is associated with microsomes. Small RNA binding is required for AGO1 stability. It would be very interesting if AGO1 is still associated with microsomes in the absence of miRNAs. The authors should add some discussions about this.2) Examine whether the mutant form of AGO1 in ago1-27 binds miRNAs by IP-sRNA northern blot and whether it is associated with microsomes or MBPs by western blot.We have included the full reviews, which mention separate points for revisions. The experiments listed in points #3, 4 and 5 of Reviewer #1 are also nice, but after discussion, we agree that these experiments are not essential for this paper. Reviewer #2 had a few minor comments, we ask you to take them into account as well.Reviewer #1:In this paper, the authors examined patterns pf subcellular partitioning of small RNAs and "big" RNAs at the genomic scale, using cellular fractionation followed by RNA deep sequencing. They found that miRNAs and their targets were associated with membrane-bound polysomes (MBPs). They also found that the AGO1 complexes purified from microsomal fraction (M) had slicer activity and 3'cleavage products could be detected in M and MBP fractions. They thus conclude that MBPs are the places where miRNA-guided cleavage occurs. More interestingly, the authors found that the phasiRNA-triggering 22-nt miRNAs and their targets including non-coding TAS transcripts were present on MBPs, suggesting phasiRNA biogenesis may be initiated on MBPs. These findings provide new insights into miRNA-guided target cleavage and phasiRNA biogenesis. Detailed comments are listed below.1) The authors used a hyl1 mutant to show that the association of AGO1 with M is mostly likely independent of miRNAs. The hyl1 mutations actually have relatively moderate effects on miRNA accumulation, compared to that caused by dcl1 mutations. I would suggest the authors use dcl1 mutant instead for this experiment. Alternatively, the authors could also examine whether sRNA binding deficient AGO1 associates with M. It would be nice to do the same experiment with MBPs.2) The authors found that the association of miRNAs with M was greatly reduced in ago1-27 suggesting that membrane association of miRNAs depended on AGO1. As ago1-27 is a week allele and the mutation has no strong effects on miRNA accumulation and slicer activity, suggesting that the mutant AGO1-27 can still bind miRNAs. The authors can test this by AGO1 IP followed by small RNA Northern blot. This is confirmed. Then the next question is how the mutation affects miRNA association with M. Does this mutation affects AGO1 association with M? This can be easily addressed by an AGO1 Western blotting with M/MBP fractions in ago1-27.3) The authors detected cleavage activity/products in M and MBP fractions. This only indicates that miRNA cleavage can occur on MBPs but does not exclude the possibility that cleavage can also occur in other places. It would be nice if the authors can try to detect cleavage activity/products in MBP-depleted fractions.4) It is very interesting that ribosome occupancy may play a role in specifying a sequence for phasiRNA biogenesis. I would suggest the authors to examine the ribosome profiles of the transcripts targeted by 22-nt miRNAs that are not capable of triggering phasiRNA biogenesis.5) It would be also interesting to examine whether the components in the phasiRNA biogenesis pathway including SGS3 and RDR6 are associated with M/MBP fractions.Reviewer #2:Post-transcriptional gene silencing can occur via transcript cleavage or inhibition of translation. Currently there is little known about the mechanism of translational inhibition in plants. This manuscript builds on previous work from the Chen lab that indicated the ER is required for microRNA-mediated translational repression. The manuscript showed differential distribution of sRNA classes in the cytosol compared with those associated with microsomes, and that microRNAs were enriched in membrane-bound polysome preparations. This membrane association in part required AGO1. Interestingly, the ago1-27 mutant, which retains AGO1 cleavage activity, showed loss of phasing of miR168-mediated target cleavage. Furthermore, and unexpectedly, TAS transcripts were found to be associated with ribosomes. Ribosome-bound regions were not uniformly distributed across these transcripts and appeared to be confined to regions upstream of the miRNA binding site. These results hint at possible new roles for membrane-bound ribosomes as a control point in sRNA production. Overall the data are novel, interesting, clear and thorough. I have only a few minor comments on the manuscript.There are two Reviewer #3:This paper is a breakthrough in miRNA regulation of plant genes. Our understanding of miRNA regulation in plants has always been more advanced than animal miRNAs, mostly because the target mRNAs for plant miRNAs are so much easier to identify and study in depth. Rather than getting confused by the poorly predicted target mRNAs of animal miRNAs, the plant field has focused on the known targets and discovered amazing biology such as the phased siRNAs triggered by plant miRNAs on their target mRNAs. This paper takes another major step by purifying mRNAs and associated siRNAs from membrane fractions and finding a dramatic difference between ER polysomes and run of the mill cytoplasmic ribosome. The difference depends on how well the authors purify the various subcellular fractions and they use various compartment specific reagents to assess their purification schemes. It is convincing fractionation and the results are dramatic. The authors demonstrate step by step that AGO1 mediates the ER association of miRNAs with mRNAs, that this association is not tied to secretion of the encoded proteins, but that AGO1 is mediating the association with polysomes. And that these miRNAS cleave target mRNAs and thus trigger the production of phased siRNAs from the miRNA phase mark. The Discussion is stunningly short and to the point. Essential revisions:1) Use dcl1 mutants to test whether miRNAs are required for the membrane association of AGO1. Alternatively, examine whether small RNA binding-deficient AGO1 is associated with microsomes. Small RNA binding is required for AGO1 stability. It would be very interesting if AGO1 is still associated with microsomes in the absence of miRNAs. The authors should add some discussions about this.dcl1-20 in the revised manuscript. The results are shown in We actually did examine, with many replications, mutants in a number of genes known to affect miRNA biogenesis. We included the results with 2) Examine whether the mutant form of AGO1 in ago1-27 binds miRNAs by IP-sRNA northern blot and whether it is associated with microsomes or MBPs by western blot.ago1-27 or they were redundant with what was shown in the manuscript. To summarize the results, the mutant AGO1-27 protein still associates with miRNAs (determined by IP followed by northern blotting), which is consistent with the result showing that AGO1 IP from ago1-27 had good slicer activity, which was presented in the original manuscript as We had done these experiments as well, but did not include the results as they were either negative results in that did not reveal how the membrane association of miRNAs is reduced in We have included the full reviews, which mention separate points for revisions. The experiments listed in points #3, 4 and 5 of Reviewer #1 are also nice, but after discussion, we agree that these experiments are not essential for this paper. Reviewer #2 had a few minor comments, we ask you to take them into account as well.Reviewer #1:[…]3) The authors detected cleavage activity/products in M and MBP fractions. This only indicates that miRNA cleavage can occur on MBPs but does not exclude the possibility that cleavage can also occur in other places. It would be nice if the authors can try to detect cleavage activity/products in MBP-depleted fractions.This is correct. We added a sentence to talk about this.4) It is very interesting that ribosome occupancy may play a role in specifying a sequence for phasiRNA biogenesis. I would suggest the authors to examine the ribosome profiles of the transcripts targeted by 22-nt miRNAs that are not capable of triggering phasiRNA biogenesis.As shown in the manuscript, many miRNAs have 22-nt isoforms, and they do not trigger phasiRNA biogenesis. We examined the MBP ribo-seq patterns of some miRNA target transcripts, and the patterns are similar to regular protein-coding genes in that the entire ORF had ribosome footprints. We did not observe evidence for ribosome stalling before the miRNA target sites.5) It would be also interesting to examine whether the components in the phasiRNA biogenesis pathway including SGS3 and RDR6 are associated with M/MBP fractions.We agree. We plan to obtain antibodies against these proteins or transgenic lines with tags for this purpose. We do not have these resources as yet.Reviewer #2:Post-transcriptional gene silencing can occur via transcript cleavage or inhibition of translation. Currently there is little known about the mechanism of translational inhibition in plants. This manuscript builds on previous work from the Chen lab that indicated the ER is required for microRNA-mediated translational repression. The manuscript showed differential distribution of sRNA classes in the cytosol compared with those associated with microsomes, and that microRNAs were enriched in membrane-bound polysome preparations. This membrane association in part required AGO1. Interestingly, the ago1-27 mutant, which retains AGO1 cleavage activity, showed loss of phasing of miR168-mediated target cleavage. Furthermore, and unexpectedly, TAS transcripts were found to be associated with ribosomes. Ribosome-bound regions were not uniformly distributed across these transcripts and appeared to be confined to regions upstream of the miRNA binding site. These results hint at possible new roles for membrane-bound ribosomes as a control point in sRNA production. Overall the data are novel, interesting, clear and thorough. I have only a few minor comments on the manuscript.We added a sentence to explain it in the legend.Thank you for this question. We neglected to mention that it is “input”.Yes, it is possible. The fractionation is not 100% clean. The fact that the 3’ cleavage fragments can be detected in the MBP fraction (after going through two sucrose gradient fractionations) adds confidence to the claim that cleavage occurs on MBP.There are two This has been corrected.


Brain cytoplasmic 200 RNA (BC200 RNA), a neuron-specific non-coding RNA, is also highly expressed in a number of tumors of non-neuronal origin. However, the biosynthesis of BC200 RNA remains poorly understood. In this study, we show that the efficient transcription of BC200 RNA requires both internal and upstream promoter elements in cancer cells. The transcription complex seems to interact with a broad range of sequences within the upstream 100-bp region. The cellular levels and half-lives of BC200 RNA were found to differ across various cancer cell types, but there was no significant correlation between these parameters. Exogenously expressed BC200 RNA had a shorter half-life than that observed for the endogenous version in cancer cells, suggesting that BC200 RNA might be protected by some limiting factor(s) in cancer cells. Transient transfection experiments showed that the transcriptional activity of the exogenous BC200 RNA promoter element varied depending on the cancer cell type. However, the promoter activities together with the half-life data could not explain the differences in the levels of BC200 RNA among different cell types, suggesting that there is another level of transcriptional regulation beyond that detected by our transient transfection experiments. Its primate homolog, brain cytoplasmic 200 RNA (BC200 RNA), was also identified in neural cells2. BC200 RNA originated from a retrotransposed Alu domain, whereas BC1 RNA arose by retroposition of tRNAAla. Primate BC200 RNA and rodent BC1 RNA are quite different in their sequences, but they share A-rich sequences in central domains. The Alu domain of BC200 RNA shares about 85% similarity to the 5′ domain of 7SL RNA3. BC200 RNA consists of a 7SL RNA-like sequence encoded by a monomeric Alu element, an A-rich central region, and a unique C-rich terminal region6. BC200 RNA is made at the cell body of a neuron and then transported to the dendrites, where it functions as a local regulator. BC200 RNA binds to various proteins, such as signal recognition particle 9/14 (SRP9/14), fragile X mental retardation protein (FMRP), poly A binding protein (PABP), and synaptotagmin-binding cytoplasmic RNA interacting protein (SYNCRIP)10. Functionally, BC200 RNA has been shown to inhibit translation by interacting with eIF4A and eIF4B12. BC200 RNA is significantly upregulated in the brains of patients with Alzheimer’s disease, suggesting that it may be involved in neurodegenerative diseases13. Although BC200 RNA was initially discovered in neurons2, it is also highly expressed in a number of tumors and cancer cell lines of non-neuronal origin17, and may be a predictive marker of prognosis in cancers. BC200 RNA has been shown to help modulate cancer cell metabolism18, which suggests that its biogenesis should be important for this regulation. Indeed, a recent study revealed that the representative onco-protein, c-Myc, activates BC200 RNA expression, and the upregulated BC200 RNA gives rise to cell metastasis in non-small-cell lung cancer (NSCLC)19.Brain cytoplasmic 1 RNA (BC1 RNA) was first identified as a poly(A)-containing non-coding RNA (ncRNA) in rat brain3. Pol III transcription has been shown to increase ncRNA transcription in cancer cells30, perhaps accounting for the high levels of BC200 RNA in some such cells. However, we previously found that BC200 RNA expression levels vary greatly across different breast cancer cell lines6. Thus, the high cellular contents of BC200 RNA in some cancer cells are not due solely to increased pol III activity, suggesting that changes in BC200 RNA stability may also contribute to the levels of this ncRNA. Furthermore, although there is a putative upstream TATA-like sequence at positions −28 to −22 and the presence of internal A and B box elements have been proposed3, the promoter elements of BC200 RNA have not yet been experimentally defined.The biosynthesis of BC200 RNA is poorly understood. The results of an α-amanitin sensitivity analysis suggested that it is transcribed by RNA polymerase III (pol III)In this study, we show that the efficient transcription of BC200 RNA requires both internal and upstream promoter elements. Our mutational analysis showed that the previously reported putative internal A and B boxes did, indeed, correspond to the internal promoter element. Our deletion analysis showed that the upstream 100-bp region is essential for the transcription of BC200 RNA in HeLa cells. We further found that the TATA binding protein (TBP) binds to the upstream 100-bp region and is required for efficient BC200 RNA transcription. The cellular levels and half-lives of BC200 RNA differed among the tested cancer cell types, but there was no significant correlation between these parameters. Finally, our results indicated that the transcriptional activity of the exogenous BC200 RNA promoter element varied across the tested cancer cell types, but the differences in promoter activity and RNA stability did not fully explain the differences in the cellular levels of BC200 RNA across different cell types. Thus, there may be another level of transcriptional regulation beyond that observed by our transient transfection experiments. Our results may provide a molecular basis for the mechanistic links between aberrant BC200 expression and tumorigenesis.Escherichia coli RNase P RNA) from the human H1 RNA promoter via the action of pol III. The expression levels of the upstream deletion derivatives relative to that of the −1010 nt construct were almost the same between BC200 RNA and RNAΔA, suggesting that the A-rich region is not required for BC200 RNA transcription and that RNAΔA can be used to monitor the contribution of the upstream sequence to the transcription rate. Deletion of sequences between positions −1010 and −100 had no significant effect on BC200 RNA expression. However, deletions from positions −100 to −1 caused the promoter to gradually and deletion-size-dependently lose its transcriptional activity. When the whole upstream sequence was deleted, the transcription dropped to less than 10% of the value from the −1010 nt construct. These results suggest that the −100 nt upstream sequence is important for BC200 RNA transcription and that the transcription complex interacts with a broad region of the upstream sequence. We thus set out to further examine this region using the −100 nt construct. Since the presence of a putative upstream TATA-like sequence (TATGAAA) at positions −28 to −22 had been previously proposed32, we used mutagenesis to analyze the importance of this sequence to the transcription of BC200 RNA. Mutation of this sequence decreased transcription to about 60% of the wild-type level  were generated for each deletion derivative. The resulting plasmids were used to transfect HeLa cells, and the exogenous expressions of BC200 RNA or RNAΔA were analyzed by Northern blotting , and two normal cell lines . We confirmed that BC200 RNA expression varied among the cancer cell lines, and that HaCaT and MCF10A cells expressed low levels of BC200 RNA  in certain cell types. The 3′ region of the Alu domain is important for maintaining the BC200 RNA stability, potentially through binding of SRP9/14 to the Alu domain35.Next, we showed that the levels of BC200 RNA in the different cell lines tested herein were not correlated with the levels of H1 RNA, a housekeeping RNA transcribed by pol III42. Human TFIIIC alleviates the chromatin-mediated repression of pol III transcription through its inherent histone acetyltransferase (HAT) ability45, and TAFII250  also has HAT activity46. Hypomethylation, especially on retrotransposable elements, is associated with genomic instability49. Recently, the transcriptions of SINEs  were shown to be inhibited by a histone methyltransferase that methylates histone H3 on lysine 951. Therefore, histone acetylation or demethylation may help activate the BC200 RNA gene in cancer cells by altering the chromatin structure.Our transient transfection experiments revealed that the BC200 RNA promoter activity varied among the different cells. The promoter activity in T47D cells was much higher than that in the non-cancer-origin cell lines, MCF10A and HaCaT. However, SK-BR-3 cells had a much lower activity than those of the non-cancer-origin cell lines. The highest and lowest promoter activities of T47D and SK-BR-3 cells, respectively, could explain why their cellular levels of BC200 RNA are high and low, respectively. However, MCF7 cells had a relatively low promoter activity but expressed the highest level of BC200 RNA with the shortest half-life among the examined cells. Therefore, the promoter activities and the half-lives do not together explain the cell type-dependent cellular levels. This suggests that there might be another mechanism responsible for the transcriptional activation of BC200 RNA. Transcription activation could be mediated by sequences far from the BC200 RNA gene fragment (positions −1010 to +300) used for transfection, and/or some alteration in chromatin structure may be essential for activating BC200 RNA transcription. Histone modifications, such as those governed by the histone acetyltransferases, GCN5 and p300/CBP, are known to activate pol III-transcribed genesIn sum, we herein show that increases in both RNA stability and transcription contribute to the upregulation of BC200 RNA seen in cancer cells. Since BC200 RNA is reported to participate in promoting metastasis, its biosynthetic regulation should be related to cancer pathogenesis. Our results provide a molecular basis for the regulation of BC200 RNA biosynthesis and improve our understanding of BC200 RNA-mediated regulatory networks in cancer metabolism.2 atmosphere at 37 °C. Normal breast MCF10A cells were grown in the same medium additionally supplemented with insulin (10 μg/ml), EGF (20 μg/ml), and hydrocortisone (0.5 μg/ml). All cell lines were found to be mycoplasma free.Cervical cancer (HeLa) cells, various breast cancer cell lines , and normal keratinocyte (HaCaT) cells were obtained from the ATCC collection. They were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% (v/v) fetal bovine serum (Gibco) and 5% antibiotic-antimycotic (Gibco) in a 5% (v/v) CO52. Constructs having 5- or 10-bp deletions within the upstream 100-bp sequence were also generated by recombinant PCR. Mutations were introduced into the A box, the B box, the putative TATA binding sequence, and nts −5 to −1, using a Muta-Direct™ Site Directed Mutagenesis Kit (Intron). For ectopic expression of BC200 RNA or its derivatives from the H1 RNA promoter, the relevant coding sequences were PCR-amplified and cloned to pSUPER (OligoEngine). The utilized oligonucleotides are presented in Table The BC200 RNA gene fragments containing nts −1010 to +300, −674 to +300, −332 to +300, −217 to +300, −118 to +300, −100 to +300, −80 to +300, −60 to +300, −40 to +300, −20 to +300, −15 to +300, −10 to +300, −5 to +300, and nts +1 to +300 were PCR amplified using Human genomic DNA (Roche) and cloned into the Tblunt vector (Solgent). Internal deletion constructs lacking an A-rich region (nts +123 to +157) and the nts +98 to +117 sequences, respectively, were generated by recombinant PCRCells were seeded, grown for 24 h, and transfected with plasmid DNA or siRNA using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The siRNA #1, siRNA #2, and siRNA #3 against human TBP  and the negative control siRNA (Cat. SN-1002) were purchased from BIONEER  was added to the media of growing cell cultures. For transfection experiments, the drug was added at 30 h post-transfection. Cells were collected at the indicated intervals after drug treatment and processed for RNA analysis. Since the actinomycin D-challenged cells started dying 8 to 16 h after the drug treatment, the data during 16 h was used to calculate the half-lives of BC200 RNA.32P] ATP and T4 polynucleotide kinase. The membrane was exposed to an imaging plate, which was analyzed with a FLA-7000 (Fuji). For semi-quantitative RT-PCR, 1 µg of total RNA was reverse transcribed with a cDNA synthesis kit (Toyobo). cDNAs were amplified using a Taq Premix kit (Enzynomics) and primer pairs specific to TBP or GAPDH .Total cellular RNA was prepared using an easy-Blue™ Total RNA Extraction Kit (Intron) according to the manufacturer’s description. For Northern blot analysis, total RNA was fractionated on a 5% polyacrylamide gel containing 7 M urea and electrotransferred onto a Hybond-N+ membrane . The membrane was hybridized with oligoprobes aBC200, aH1, a5S, and aM1 Table  that hadCells were lysed in RIPA buffer . The cell lysates were resolved by SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes . Immunostaining was carried out with polyclonal rabbit anti-TBP antibody  using the Amersham ECL Prime Western Blotting Detection Reagent  according to the manufacturer’s instruction.54, and chromatin immunoprecipitation (ChIP) was performed. Briefly, a solution containing 2 mg of sheared formaldehyde-crosslinked-chromatin was incubated with rProtein G-agarose beads  for 2 h at 4 °C for pre-clearing. The supernatant fraction was incubated with 10 µg of polyclonal rabbit anti-TBP antibody overnight at 4 °C, and then with rProtein G-agarose beads for 2 h at 4 °C. The beads were washed with RIPA buffer  and the DNA was eluted with elution buffer . The isolated DNA was PCR-analyzed using BC200 RNA or GAPDH gene-specific primers (Table Chromatin was cross-linked with formaldehyde as previously describedrs Table .Supplementary Data


Arabidopsis thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from  Expression of the chloroplast genome is the result of a complex interplay of nuclear and organelle-encoded proteins. As many as three RNA polymerases and six sigma factors create the primary transcriptome, which is further processed by an array of ribonucleases, RNA-binding proteins, and RNA splicing and editing factors, ultimately producing mature transfer RNAs, ribosomal RNAs, and messenger RNAs  .The first global strategies for chloroplast transcript analysis utilized microarrays, most recently tiling arrays, which have yielded breakthroughs in our understanding of RNA targets of specific regulatory factors, developmental variations in the transcriptome, and more recently translation , SAMtools (http://www.htslib.org/), TopHat2 version 2.0.11 (http://ccb.jhu.edu/software/tophat/index.shtml), Cufflinks version 2.2.1 (http://cole-trapnell-lab.github.io/cufflinks/), and BED tools version 2.25.0 (https://github.com/arq5x/bedtools2).All analyses described here were performed on a 64-bit laptop running the Ubuntu 14.04 LTS operating system with 8 GB (gigabytes) of RAM, with the following software installed: Bowtie2 version 2.2.3 (http://www.ncbi.nlm.nih.gov/sra?term=SRP028304), and then converted into the corresponding FastQ file using fastq-dump from the SRA toolkit (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc&f=fastq-dump). Reads were initially quality trimmed using the fastq-mcf software (https://code.google.com/p/ea-utils/wiki/FastqMcf), and only bases with a quality score higher than 30 and reads with a minimum length of 60 were retained.RNA-Seq data  and ungrouped in the working directory. The annotation files specify genomic intervals to be used by ChloroSeq (see below). The TAIR10_ChrC_files directory contents further expand in TAIR10_ChrC_bowtie2_index, and contain the indexed files for the chloroplast genome used during the alignment.Before starting, ChloroSeq and the accompanying annotation files must be downloaded . The GFF file containing the gene coordinates and the indexed genome files used can be found in the TAIR10_ChrC_files and TAIR10_ChrC_files/TAIR10_ChrC_bowtie2_index directories, respectively, and the novel splice site discovery was disabled (–no-novel-juncs).File S1) containing the genes known to be involved in chloroplast RNA metabolism, to simplify the extraction of their expression values.In addition, differential expression of nuclear genes was assayed following the step-by-step Tuxedo protocol using TopHat and Cuffdiff  that prepare the read file for processing, then address coverage, splicing, and editing, and one Perl script (.pl file) that runs the pipeline and calls the bash shell scripts as needed. All of these files need to be made executable before use. Here is an overview of ChloroSeq:perldoc chloroseq.plWhen the pipeline is first run, the shell script prep_bam.sh is called to index the bam file. The desired reads corresponding to the genome of interest (here ChrC) are then extracted from the aligned reads bam file. The resulting bam file is then used for all other steps in the pipeline. This gives ChloroSeq the capacity to extract and analyze any subgenome of interest (for example chloroplast or mitochondrion) when the alignment has been performed against the complete genome. One simply needs to know the size of the genome (-g option) and the name of the genome of interest (-n option). The name of the genome of interest must match the one used for alignment.C is a constant (C = 109), Ri is the number of reads that intersect the region of interest, Ti is the total number of reads of the sample, and L is the length of the region.By choosing option analysis 1 (-a 1) in the chloroseq.pl pipeline, the get_coverage.sh script is called and produces four different outputs: (1) single nucleotide depth of coverage of the complete chloroplast genome, (2) genome coverage using a 100 nt window, where each window overlaps the next by 50 nt, and RPKM values for (3) exons and (4) introns. RPKM values are calculated according to the formula Once completed, six text files containing the four different outputs are found in the working directory: counts, bam_name, nt_coverage.txt, window_coverage.txt, exon_rpkm.txt, and intron_rpkm.txt. Intermediate files created may be kept with the –k option and are named coverage_plus.csv, coverage_minus.csv, window_plus.csv, window_minus.csv, exp_exon.txt, and exp_intron.txt.SE) was calculated according to the formula jR is the number of reads spanning the exon/exon junction, liR is the number of reads spanning the 5′ exon/intron boundary, and riR is the number of reads spanning the 3′ intron/exon boundary.Analysis 2 calls the get_splicing_efficiency script and produces one output file that contains the coordinates, the number of spliced and unspliced reads, and the calculated splicing efficiency for each intron. Splicing efficiency  according to the manufacturer’s instructions. For RNA blot analysis, 1 or 5 µg of total RNA was separated in 1.2% agarose/formaldehyde gels, blotted overnight onto Hybond-N+ , and hybridized with either α-32P-dCTP-labeled double-stranded DNA or α-32P-UTP-labeled single-stranded RNA probes as previously described . All bioinformatic scripts developed for this project can be found on GitHub (https://github.com/BenoitCastandet/chloroseq).The RNA-Seq data used in this article was obtained from the SRA database , a pipeline designed to reveal these subtler, yet critical facets of chloroplast gene expression. The strategy is depicted in A. thaliana genome, modified to add the first exon of the chloroplast gene ycf3. ChloroSeq has been optimized for single-end strand-specific RNA-Seq data, but can be adapted to work with paired-end reads. Additional details are given in Materials and Methods.Most published RNA-Seq libraries have only been used to study differential gene expression (DGE), and step-by-step protocols have been published to facilitate such analyses  RNA using oligo(dT) to prime cDNA synthesis, or deplete rRNA through hybridization to complementary oligonucleotides. Only the latter method preserves chloroplast transcripts, which are primarily free of poly(A) tails, and was applied to nearly 200 plant RNA-Seq libraries deposited in the SRA database. rRNA subtraction is becoming more widely used, however, because of the recognition that important components of the nuclear transcriptome are not polyadenylated, such as noncoding RNAs (ncRNAs)  RNA specifically regulated by chloroplast Mini-ribonuclease III, and previously unidentified 3′ extensions and cleaved tRNA leaders found in the PNPase mutant  window coverage overlapping by 50 nt, normalized to the total number of reads. Graphical representation of the window coverage, for example, can be used for quick identification of regions that merit further investigation or validation using molecular techniques. Additionally, a more precise view, for instance where coverage varies dramatically within a region, can be achieved using the single nucleotide coverage output. Importantly, either coverage analysis does not require any A. thaliana plants grown under control, heat, salt, cold, and drought stress conditions . Figure S1 illustrates how the complete genome can be rapidly scanned by eye to reveal relative expression levels, while In the present study, ChloroSeq 100 nt coverage was completed using RNA-Seq data from Figure S1A). In the heat map, many areas outside the genic regions (light gray bars at edges of Brassica rapa (vs. 37°), type of pncRNA detected , and species examined, but rather corroborates the influence of heat on pncRNA accumulation. To see whether apparent pncRNA increases would be reflected in RNA gel blots, we examined transcripts antisense to atpH . Only after 12 hr of heat stress did total exonic RPKMs appear higher than the control condition, but the difference is not statistically significant . However, this trend is amplified for introns, for which median RPKMs are approximately fivefold higher after 12 hr of heat stress compared to control and other stress conditions . Examination of individual exons and introns revealed that at 12 hr of heat stress, a small number of exons appeared to be responsible for the overall RPKM increase . This is well illustrated by the atpF intron, for which four proteins  . Therefore, the results to date do not support a direct link between expression at the RNA level of a given splicing factor and splicing efficiency.Akin to mechanisms underlying altered RNA accumulation, splicing nd CRS1) . This repsbF_63985, psbE_64109, ndhD_116785, ndhB_95225, or rpl23_86055), or modest increases   . Differeor rps14  also occor rps14 . Site-sp tobacco , 2002, a tobacco , is incrbidopsis .rpoB_25992 displays a stronger reduction after 3 hr of heat stress compared to 12 hr, and the rps12_69553 site experiences an increase in editing after 3 hr and a decrease after 12 hr. It is worth noting that two recently identified proteins involved in chloroplast RNA editing, RIP2 and RIP9 . In some cases, editing factor expression correlates with editing efficiency . For example, CRR21 and OTP80 expression remained stable under heat stress, as did their corresponding editing sites ndhD_116785 and rpl23_86055. On the other hand, editing sites rps14_37161, clpP_69942, and rpoA_78691 displayed a large reduction in editing, while expression of their editing factors OTP86 and CLB19 increased or remained stable, respectively. Finally, editing at ndhD_117166, which creates the AUG initiation codon of the ndhD transcript, is strongly inhibited under heat stress. Its editing requires the cooperative association between CRR4 and DYW1 (Some sites show a different behavior. Editing at We have presented ChloroSeq, a bioinformatics pipeline developed to investigate the chloroplast transcriptome systematically utilizing RNA-Seq data. Using ChloroSeq, information on chloroplast transcript accumulation, splicing, and editing can be determined simultaneously, and can be combined with nuclear transcript analysis. Although not shown here, ChloroSeq can also be used to study the mitochondrial transcriptome, which features some of same RNA-Seq analytical challenges as the chloroplast . MoreovePhaseolus vulgaris (Here, we demonstrated the power of ChloroSeq through analysis of previously published datasets on abiotic stress, for which organellar data had not been studied. Among the stresses, heat stress was specifically associated with multifaceted effects on the chloroplast transcriptome. These included increased pncRNA accumulation, and decreases in splicing and editing efficiencies, all of which are phenomena that could potentially impact chloroplast protein synthesis. A global decrease in chloroplast protein synthesis following heat stress was earlier observed in vulgaris . The mai