Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5′ UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite  Leishmania major  lengths for a particular gene  binding proteins as well as four isoforms of the translation initiation factor (eIF4E) and five eIF4G isoforms . Moreovelar gene ,35,36. Tlar gene . Such relar gene . uORFs clar gene ,40. Baseone uORF .T. brucei  have been successfully used to estimate the rates of translation (rates of protein synthesis)  . Ribosomnthesis) .Importantly, this approach not only provides quantitative information on the number of RNA molecules associated with ribosomes, it also yields position-specific information regarding the location of ribosomes on mRNA transcripts. This information is critical, as association of an RNA transcript with a ribosome does not necessarily mean that the transcript is being translated. The ribosome could be associated with the 5′ UTR or it could be stalled, failing to produce functional protein. In addition, ribosome positions can be used to accurately map the correct CDSs and help identify novel CDSs that have been missed in previous genome annotations .T. brucei. Comparative ribosome profiling analyses of the bloodstream and procyclic parasite forms allowed us to generate a genome-wide picture of translation. We observed a 100-fold range in translational efficiency among genes and life cycle-specific differences in translational efficiency for a large number of genes. In addition, our ribosome profiling data enabled the identification of hundreds of previously un-annotated CDSs and incorrectly annotated translation initiation sites, and suggest a regulatory role of uORFs.Here we report the adaptation of a ribosome profiling approach to T. brucei strain Lister 427 were cultured at 27°C in SDM-79 supplemented with 10% fetal bovine serum and hemin (7.5 mg/l) medium (7 cells/ml. Wild-type bloodstream form (BF) of Lister 427 (MITat 1.2 clone 221) were cultured at 37°C in HMI-11 up to a cell density of 1.5 × 106 cells/ml. Two minutes before harvest, cycloheximide was added to a final concentration of 100 µg/ml. Cells were collected by centrifugation at 3000 × g, 4°C for 5 min, washed with polysome lysis buffer , transferred to a 1.5-ml microcentrifuge tube and pelleted. To lyse the cells, 360 µl polysome lysis buffer, 40 µl of 10% n-octylglycoside and 20 units of TURBO DNaseI (Ambion) were added per 109 cells and incubated for 30 min on ice. The lysate was centrifuged at 16 000× g, 4°C for 10 min, the supernatant transferred to a new microcentrifuge tube and the OD260 was determined using a Nanodrop 2000.The procyclic form (PF) of ) medium  up to a 260 = 40) were digested with RNase I (Ambion) at RT (1200 units) or on ice (1600 units). After 1 h, the digestions were stopped by adding 100 units of SUPERase•In RNase inhibitor (Ambion) to the RNase-treated samples. In parallel, 100 units of SUPERase•In RNase inhibitor were added to a 200 µl aliquot of lysate not containing RNase I (undigested control). Monosomes were purified using sucrose gradients as described previously  at 65°C . To geneSequencing libraries were prepared as described  except fhttp://code.google.com/p/cutadapt/) and reads shorter than 20 nt after trimming were removed. Trimmed reads were mapped to the reference genome using bowtie-2 with default ‘local-sensitive’ mode  was trimmed from the 3′ end of reads using cutadapt . Non-structural RNA refers to the reads mapped to the genome excluding annotated rRNA and tRNA regions. Translational efficiency of a region was defined as its ratio of abundance in the ribosome profiling library to that of the control RNA library of the same life cycle stage. To investigate the distinct 3-nt periodicity of the ribosome profiling libraries, 5′ ends of the mapped reads were piled up and the piled-up coverage of each position from each gene (using either start or stop codon as the reference point) were summed up in meta-gene analyses. Genes with less than 10 reads within the plotted region were excluded.A uORF was defined as any ORF ≥9 nt that was located within an annotated 5′ UTR and was on the same strand as the annotated ORF. Novel CDSs are defined as any translated ORF ≥ 30 nt located at least 20 nt away from any existing annotations. In cases in which multiple overlapping novel ORFs were found on different frames of the same strand, only the longest were retained. An ORF is defined as translated when ≥ 70% of its CDS is being covered by the pooled ribosome profiling reads (i.e. four libraries) with ≥2 reads per nucleotide.rpk and pUTR3rpk) in both ribosome profiling and RNA-sequencing control libraries were calculated for each ORF. The ratio of CDSrpk to pUTR3rpk in both ribosome profiling and RNA-sequencing control libraries (i.e. riboratio and RNAratio) were calculated. RRS of each ORF was then defined as the ratio of riboratio to RNAratio.Ribosome release scores (RRSs) of all ORFs, including annotated CDSs, putative CDSs and uORFs, were calculated as described with slight modifications . Brieflyhttp://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) and EdgeR (P < 0.05 in each case) linear fold change ≥ 5 were considered to be potentially related to the loss of fitness. The pair-wise library comparisons include BF uninduced versus BF induced for 6 days, BF uninduced versus differentiated cells induced and BF uninduced versus PF induced.We reanalysed the data of a published genome-wide RNA interference fitness–costs association study  with ournd EdgeR , with deet al. , a list of uORFs and putative CDSs identified in this study (Supplementary Tables S4 and S5) and a database containing typical contaminants. Enzyme search specificity was Trypsin/P with three miscleavages for tryptic digests and LysC with two miscleavages for digests with lysyl endopeptidase. Carbamidomethylation on cysteines was set as fixed modification; methionine oxidation and protein N-terminal acetylation were considered as variable modifications. Mass accuracy tolerances  were 6 ppm for precursor ions, and 0.5 Da for CID MS/MS and 20 ppm for HCD MS/MS spectra, respectively. False discovery rate was fixed at 1% at peptide and protein level, with posterior error probability (PEP) as sorting criterion. Identifications were matched between runs within a 2-min window. Protein groups identified in the potential CDS database had at least one unique peptide and a PEP < 0.0005.All mass spectrometric raw data files from Butter et al.  were proh engine ,59 was uh engine . The seaT. brucei. We sequenced ribosome footprints and fragmented mRNA from the two trypanosome stages that have been adapted to in vitro culture, the PF of the tsetse fly midgut and the proliferating BF that lives in the blood of the mammalian host. Cultured cells were treated with cycloheximide to arrest translating ribosomes and harvested. Because cycloheximide has been shown to stabilize RNA transcripts in T. brucei . Next, to map translated regions of RNA and to exclude RNase-protected regions that are scanned by the 43S pre-initiation complex, we used sucrose gradients to specifically enrich for monosomes . The high reproducibility between libraries prepared under different conditions demonstrated the robustness of the ribosome profiling approach despite the elaborate library preparation protocol .To obtain genome-wide information on protein synthesis, we adapted a ribosome profiling protocol published in yeast and mice ,46 for u. brucei , the treSupplementary Figure S3). No such 3-nt periodicity was observed for the mRNA sequence reads.To ensure that nuclease-resistant RNA fragments represent ribosome footprints and not short RNA fragments protected by other RNA-binding proteins, we compared the genome-wide distribution of sequence reads obtained from ribosome profiling and RNA-sequencing libraries. Alignment of ribosome profiling reads revealed that the nuclease-protected reads predominantly mapped along CDSs with very few reads aligning to intergenic regions A. The 5′T. brucei genes have introns, one encoding a poly(A) polymerase and the other encoding a DNA/RNA helicase , i.e. the 5′ end of the 5′ UTR to which the spliced leader RNA is ted CDSs ,35,36. TIn contrast to RNA-sequencing, ribosome profiling allows the determination of the translational landscape. Thus, it allows the identification of CDS mis-annotations and the determination of the most probable translation initiation sites. Exemplary for numerous genes, for Tb927.5.1990 we detected no translation between the first and the second AUG (green bars) indicating that this region is not translated in the BF and PF and that translation initiates at the second AUG , see ‘Materials and Methods’ section]. Of 77 million PF and 44 million BF ribosome footprint sequence reads, 28% (PF) and 14% (BF) could be aligned to annotated CDSs, while 70% (PF) and 85% (BF) mapped to known structural non-coding RNA (ncRNA). The high percentage of structural ncRNA in the footprint fraction has been reported previously  and resuSupplementary Table S1). We found the rate of translation to differ greatly among proteins. In the PF, we observed a 11 448-fold difference in ribosome density (3548-fold difference in mRNA levels) between the 1% most highly and the 1% most weakly translated proteins, while in the BF we observed a 1623-fold difference in ribosome density and a 262-fold difference in mRNA levels , we were able to detect ≥ 10 ribosome footprint reads, a threshold that we defined as translation . Nevertheless, whenever determining the translational efficiency we excluded mRNA and footprint reads that mapped to the first 40 nt of a CDS to avoid possible artifacts from ribosome stalling at translation initiation sites. Between the 1% most efficiently and the 1% least efficiently translated proteins, we observed in the PF a 117-fold and in the BF a 64-fold range in translational efficiency, i.e. in the amount of proteins produced per transcript (Supplementary Table S2). This range in translational efficiency is similar to what has been observed in yeast and roughly 10-fold higher than what has been described for mice (2 = 0.03), suggesting that translational efficiency is regulated independently of RNA stability.To determine the translational efficiency for individual genes, we normalized for differences in mRNA abundance, i.e. we divided the ribosome footprint density by the levels of mRNA abundance. Our ribosome profiling data indicate a relatively unbiased distribution of ribosome density across CDSs in the BF and a very minor increase in density towards the 5′ end of CDSs in the PF , but it also revealed distinct life cycle-specific differences for subsets of genes. For example, 58 genes found in the lowest 25% in terms of translational efficiency in the PF were among the top 25% most efficiently translated proteins in the BF. These contain various RNA binding proteins, numerous phosphatases and expression site-associated genes (Supplementary Table S3). For an example of a gene more efficiently translated in the BF than in the PF see Next we addressed the question if translational efficiency is regulated in a life cycle-specific manner. Ribosome density measurements only allow meaningful comparisons of the rate of protein synthesis when the speed of translation is assumed to remain constant. This assumption is supported by measurements in mouse embryonic stem cells ,64; howePlasmodium falciparum, T. brucei, humans and yeast (Supplementary Table S2).Based on mRNA and protein level measurements, differential translational efficiency has been predicted for aconitase, the cytochrome oxidases V and VI and procyclins ,25,65. Fnd yeast . InteresT. brucei.Given the large dynamic range and the life cycle-specific regulation, we propose that differences in translational efficiency contribute substantially to the control of gene expression in T. brucei, sequence motifs in the 3′ UTR have been linked to developmentally regulated changes in translational efficiency, but for the large majority of genes no such motifs have been identified . Thus, uORF are much less common in T. brucei than in mammals where 40–50% of genes contain uORFs (T. brucei.Even though 5′ UTRs have only been assigned for 4909 genes (927 version 4.2), applying the criteria listed above, we identified 8310 uORFs and found 1092 (22%) of 5′ UTRs to contain at least one uORF  with ≥ 2× read-coverage and ≥70% of the ORF covered. The average length of these uORFs was 22 aa. Just like for annotated CDSs, the alignment of ribosome profiling reads across these uORFs revealed a distinct 3-nt periodicity, with 63% of BF ribosome profiling reads starting at the first nucleotide of a codon A and B. N = 1092) and those without uORFs (N = 3817). In the BF, median ribosome density across the CDSs of transcripts with uORFs was 30.31 rpkm compared with 44.86 rpkm for genes without uORFs (P < 0.0001). While we also observed a higher mRNA level for genes without uORFs compared with genes with uORFs (P < 0.0001), the differences in mRNA levels were lower than the differences in ribosome density . For 5′ UTRs we found a higher ribosome density in the PF (median rpkm: 7.73) than in the BF . Unexpectedly, the contrary was true in PF. In the PF we found 5′ UTRs without uORF (8.34 rpkm) to have a higher ribosome density than 5′ UTRs with uORF . For an example of a non-AUG uORF see Noteworthy, while median ribosome density for CDSs was higher in the BF than in the PF , we obsetrans-splicing  have been shown to play major roles in plant and animal development ,78, the Supplementary Table S5). For 797 of the 1114 previously identified transcripts, we found the average ribosome footprint read-coverage to be ≥2× (Supplementary Table S6. Previously identified transcripts from chromosome 10 were not considered because our sequencing reads were mapped to a newer version of the chromosome 10 assembly and thus genomic coordinates might be incompatible.).We mapped ribosome footprint reads from the BF and PF and determined the ribosome footprint density for all potential CDSs at least 10 aa in length. To avoid overlap with annotated CDSs, we only considered ORFs located at least 20 nt away from annotated features. This approach enabled us to identify a set of 2021 candidate CDSs with ≥2× read-coverage and ≥70% of the ORF covered. The candidate CDSs ranged in length from 10 to 378 aa . We suspect that the low number of identified protein products relative to the large number of candidate CDSs may be caused, (i) by the experimental set-up of the original proteomics analysis where proteins were separated by 1D-SDS-PAGE prior to in-gel protease digest and mass spectrometric analysis whereby proteins with a size smaller than 5–10 kDa typically run out of the gel, (ii) by the limited sensitivity of mass spectrometric analyses compared with DNA sequencing-based techniques and (iii) by the fact that not all ribosome-protected RNAs are translated.To determine whether our set of candidate CDSs is translated, we analysed published proteomics datasets  to searcThe first point is supported by the fact that only 67 (3%) of our candidate CDSs were ≥90 aa (≥10 kDa) but for 28% of those large candidate CDSs we could identify peptides. Evidence supporting the possibility that not all ribosome-protected RNAs are translated exists in mice where even known long ncRNAs have been found to be associated with ribosomes . To evalT. brucei (T. brucei (Supplementary Table S7). For these 1445 genes the median RRS was 83.9 compared with an RRS of 0.19 for the subset of genes annotated as hypothetical unlikely (N = 90). Thus, similar to the observation in mice, the RRS appears to be a good predictor of productive translation. Next we determined the RRS for the previously un-annotated candidate CDSs that fulfilled the criteria for RRS calculation (N = 265). The median RRS of these candidate CDSs was with 3.99 lower than the median RRS of the annotated genes (83.9). Thirteen candidate CDSs had an RRS larger than 83.9 and 98 candidate CDSs had an RRS higher than the well-described intron-containing poly(A) polymerase (RRS = 9.63). The RRS could only be calculated for one of the candidate CDSs for which we had identified protein products (RRS = 57.44).Similarly to annotated CDSs and uORFs, our averaged ribosome footprint data indicated a well-defined 3-nt periodicity for sequence reads occurring near translation initiation sites. However, we observed a less well-defined drop in ribosome densities across translation termination sites A and B, . brucei A and B, . brucei , i.e. thSupplementary Table S5), no motifs were identified.Finally, in an attempt to learn about the function of the putative CDSs, we searched for known protein motifs in those candidate CDSs for which we had identified protein products and for those with an RRS above 10. However, except for one candidate CDS located within the histone H2B gene array and which contained an H2B signature motif  suggest extensive translation beyond the annotated CDSs in The combination of ribosome profiling data with previously published mass spectrometric data allowed us to identify a large number of new CDSs. Nonetheless, the biological significance of these CDSs remains to be determined. In addition, while ORFs with a high ribosome footprint density, a low RRS and without mass spectrometric evidence may not be translated into functional proteins, they may, nevertheless, play important regulatory roles.Supplementary Table S8). For examples of candidate CDSs potentially essential for viability in the BF see Therefore, we decided to evaluate the importance of our candidate CDSs, independent of RRS and mass spectrometric evidence, in parasite survival. A previously published high-throughput phenotyping approach, termed RIT-seq, measured the fitness–cost associations using RNA interference (RNAi). The RIT-seq data revealed a significant loss in fitness on RNAi induction of 2724 CDSs in the BF, 1972 CDSs in the PF and 2677 CDSs in induced and differentiated parasites . The appT. brucei. It is the first such analysis for a eukaryotic pathogen and an organism without transcriptional control. Our data reveal large differences in translational efficiency among transcripts in the same life cycle stage and between the same transcript in different stages. In addition, the sequencing of ribosome-protected RNA enabled us to identify transcripts that are likely to be translated. Hundreds of putative, previously unidentified CDSs appear to be essential for parasite fitness, and the analysis of available proteomics data confirmed the existence of at least 20 of these previously unknown CDSs.In this study, we report the first genome-wide analysis of protein synthesis and a strand-specific analysis of RNA transcript levels for T. brucei. Under a single condition, we observed translational efficiency to vary over two orders of magnitude; thus, its importance equals that of RNA stability for which a similar range has been measured  of the University of Wuerzburg, Germany; German Research Foundation DFG [SI 1610/2-1]; Human Frontier Science Program (to T.N.S.); French National Research Agency . Funding for open access charge: German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing.Conflict of interest statement. None declared.


A-to-I RNA editing is a widespread post-transcriptional modification event in vertebrates. It could increase transcriptome and proteome diversity through recoding the genomic information and cross-linking other regulatory events, such as those mediated by alternative splicing, RNAi and microRNA (miRNA). Previous studies indicated that RNA editing can occur in a tissue-specific manner in response to the requirements of the local environment. We set out to systematically detect tissue-specific A-to-I RNA editing sites in 43 human tissues using bioinformatics approaches based on the Fisher's exact test and the Benjamini & Hochberg false discovery rate (FDR) multiple testing correction. Twenty-three sites in total were identified to be tissue-specific. One of them resulted in an altered amino acid residue which may prevent the phosphorylation of PARP-10 and affect its activity. Eight and two tissue-specific A-to-I RNA editing sites were predicted to destroy putative exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs), respectively. Brain-specific and ovary-specific A-to-I RNA editing sites were further verified by comparing the cDNA sequences with their corresponding genomic templates in multiple cell lines from brain, colon, breast, bone marrow, lymph, liver, ovary and kidney tissue. Our findings help to elucidate the role of A-to-I RNA editing in the regulation of tissue-specific development and function, and the approach utilized here can be broadened to study other types of tissue-specific substitution editing. RNA editing is a widespread post-transcriptional modification mechanism that alters genetic information at the RNA level by nucleotide insertions, deletions or substitutions, which can contribute to the diversification of the transcriptome and proteome Nucleotide substitution of RNA editing can change the amino acid sequence, or create or destroy the translation initiation or termination codon. Nucleotide insertion or deletion from RNA editing can result in a translational frameshift that creates new open reading frames. The consequences of these editing events can increase the repertoire of available proteins Sorghum bicolorPrevious studies have shown that some RNA editing events are tissue-specific and play important roles in physiological processes. More than 100 C-to-U substitutions in grape mitochondria were shown to be tissue-specific and may contribute to different tissue requirements Although tens of thousands of A-to-I RNA editing events have been found in humans by computational and experimental methods, there is limited knowledge of its tissue-specificity in humans. To fully understand of this type of editing event, it is necessary to perform large-scale discovery and characterization of tissue-specific A-to-I RNA editing events. The methods based on expressed sequence tags (ESTs) for large-scale analysis of tissue specificity have been successfully used to study gene expression Redundant records of the previously identified A-to-I RNA editing sites Following strict filters described in the An RNA editing event happens after gene transcription. Therefore, the expression profile of a gene limits the possibility of an RNA editing event. To test whether high expression of a gene in a tissue could increase its RNA editing level in the same tissue or not, we investigated the tissue-preferred expression of genes which contain tissue-specific RNA editing sites. By searching the Tissue-specific Gene Expression and Regulation (TiGER) database, we found that the CXCL12 gene with a connective-tissue-specific A-to-I RNA editing site in its 3′-UTR (or intron in other isoforms) was preferentially expressed in soft tissue, heart and spleen. CXCL12 can activate lymphocytes and take part in the metastasis of prostate cancer On the other hand, we analyzed the tissue-preferred expression of all annotated 2,040 genes with 18,616 A-to-I RNA editing sites. Three hundred and seventy-eight of these genes were expressed in a tissue-specific manner according to the TiGER database collection. Except CXCL12 as mentioned above, there was only one muscle-specific gene, SYNPO (an actin-associated protein), with an adipose-tissue-specific A-to-I RNA editing site. This observation indicated that the vast majority of tissue-preferred genes in this study did not contain putative tissue-specific A-to-I RNA editing sites.Therefore, we concluded that the A-to-I RNA editing and the expression of the corresponding editing substrate did not show the same tissue preferences in our study. That is, high expression of a gene does not increase its RNA editing level, and the tissue-specific editing can exist in transcripts that are widely expressed.To experimentally validate the predicted brain-specific and ovary-specific editing sites, two human tissue samples (brain) and ten human cell lines  were used. We sequenced matching DNA and RNA samples retrieved from the same specimen. As shown in Some A-to-I RNA editing sites are located in protein-coding regions, whereas the majority is found in non-coding regions. An editing site within the protein-coding region of an mRNA can result in a sequence change that may lead to an amino acid alteration in the protein. By analysis using EditFunc, one blood tissue-specific RNA editing site was found to alter an amino acid residue. The editing site mapped to chr8_−_145130527 changes the serine residue at position 507 of the PARP-10 protein (Genbank accession: NP_116178) to a glycine residue, which was predicted as a putative phosphorylation site by the EditFunc web server with the use of the NetPhos software PARP-10 belongs to the family of Poly (ADP-ribose) polymerases, which regulates gene transcription by altering chromatin organization by adding ADP-ribose to histones. PARP-10 was reported to interact with the Myc protein and inhibit cell proliferation In recent years, some evidences have accumulated showing that splicing and editing can influence each other RNA editing is an important post-transcriptional regulation that can increase protein diversity and enrich the regulation of non-coding RNA. Although a few studies have indicated that RNA editing is an indispensable modulation in response to the requirements of specific cell types, it has been a challenge to gain an overview of the global landscape of tissue-specific editing. In this study, we successfully detected human tissue-specific A-to-I editing sites by statistically analyzing EST/mRNA sequences. The overwhelming majority of the known RNA editing sites used here was found in the non-coding sequences, and most of the predicted editing sites were located in the non-coding regions as well. By gaining a deeper understanding of the non-coding sequences, we should begin to know more about the functions of the tissue-specific RNA editing.Interestingly, most of the genes containing the tissue-specific A-to-I RNA editing did not exhibit tissue-specific expression. On the contrary, many tissue-specific genes were not discovered to have the predicted tissue-specific A-to-I RNA editing sites, although we could not exclude the possibility that they may have other unknown tissue-specific RNA editing sites. This implies that the tissue-specific editing event is a modulatory mechanism required for tissue-specific development but that its role is independent of the regulation of tissue-specific gene expression. The members of the family of ADARs are the only enzymes that are known to regulate A-to-I RNA editing levels. However, it seems that the regulation by ADARs cannot completely explain how tissue-specific editing occurs. Recent studies indicated that editing levels can increase or decrease with a constant (or not significantly changed) protein expression of ADARs RNA-seq data can also be used to detect tissue-specific editing if the read sequences are treated as EST/mRNA sequences. However, the high expense of whole genome and transcriptome sequencing currently restricts its application for RNA editing analysis, and there are only three published works that have utilized high-throughput sequencing to detect RNA editing at present Finally, we have to note that there are probably many more tissue-specific editing sites than those identified in this work for the following reasons. (i) The coverage of expressed sequences in the same editing sites in all tissues are not equivalent. Therefore, many editing sites may be detected in a only few tissues but not in others where there are just too few or no expressed sequences. (ii) The Fisher's exact test with the Benjamini & Hochberg correction is usually considered strict and may cause us to miss detection of some true tissue-specific editing sites. (iii) Finally, many A-to-I RNA editing sites have been uncovered to date, and the 23 tissue-specific A-to-I RNA editing sites predicted here still represents a small portion of the actual tissue-specific RNA editing repertoire. Nevertheless, this is the first study to explore tissue-specific A-to-I RNA editing in humans, and the information gained here may facilitate the understanding of regulation by RNA editing related to the unique functions of tissues.Five sources of data were required for our analysis, including known A-to-I RNA editing sites, the human reference genomic sequences, the human mRNA/EST sequences, the alignments between the human mRNA/EST and reference genome sequences, and the human mRNA/EST library information. The total of 32,316 non-redundant A-to-I RNA editing sites identified by different methods were collected from four published works Four hundred and ninety cDNA libraries with tissue annotations were downloaded from the UCSC website. A total of 111 cDNA libraries were excluded from the original set because these libraries lacked clear tissue source information or were from mixed tissue samples. Furthermore, libraries recorded as having the same tissue source (e.g. ‘brain’) were combined into a single category, including both normal and cancerous samples from the same tissue. Finally, we filtered and grouped 379 cDNA libraries into 43 unique tissue types .As a measure of tissue-specificity, Fisher's exact test was applied to assess the significance of different RNA editing levels in all tissues, and the Benjamini-Hochberg method was used to estimate the total FDR in each tissue for correction of multiple testing.For each RNA editing site P values were ranked in ascending order P value that met the requirement, its corresponding null hypothesis and all those having smaller The following simple procedure to control the FDR at level To explore whether genes containing the A-to-I RNA editing sites were expressed in a tissue specific manner or not, we searched their expression profiles from the TiGER database Two brain adjacent non-cancerous tissue samples and ten cell lines were used in this study for experimental validation. The brain tissue samples were obtained from the 307 Hospital of PLA with the written informed consent of patients and with approval for experiments from the ethics committees of the hospital and the Beijing Institute of Biotechnology. The human glioma cell line SF126 was purchased from the Cancer Institute and Hospital, Chinese Academy of Medical Sciences (CAMS). The two human ovarian cancer cell lines (SKOV3 and OVCAR3) were supplied by Peng Peng . The human colon cancer cell line SW480, the human estrogen receptor (ER)-negative breast cancer cell line MDA-MB-231 and the human osteosarcoma cell line U2OS were supplied by Xuemin Zhang . The human chronic myeloid leukemia cell line K562 and the human Burkitt's lymphoma cell line Raji were gifts from Qingfeng Du . The human renal epithelial cell line 293T and the human hepatoma cell line HepG2 were supplied by Yan Lin .2 incubator with humidified air.K562, SW480, SKOV3 and Raji cell lines were cultured in RPMI 1640 media (Gibco) supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 0.1 mg/ml streptomycin (Hyclone). HepG2, MDA-MB-231, OVCAR3, U2OS and 293T cell lines were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. SF126 was maintained in Minimum Essential Medium (MEM) (Gibco) with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin. All cells were cultured at 37°C in a 5% COFor experimental validation of brain and an ovary tissue-specific RNA editing sites, total RNA and gDNA of two brain tissue samples isolated from the same specimen and ten cell lines were processed using standard protocols for reverse transcription and PCR. To remove genomic DNA contamination, RNA samples were treated with DNase I . First-strand cDNA was synthesized from the total RNA with the Transcriptor High fidelity cDNA Synthesis Kit (Roche) using random primers. Using the cDNA and gDNA as templates, PCR was performed according to standard procedures with 30 pM of each primer and 2.5 U rTaq DNA polymerase  to amplify the edited transcripts and the genomic DNA. The cycling conditions for amplification were as follows: initial denaturation at 95°C for 5 min, then 30 cycles at 95°C for 30 s, 59°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 10 min. Control experiments were conducted without the reverse transcriptase enzyme added (no RT control) to verify that the amplified products were from the reverse transcribed mRNA and not from contaminating genomic DNA. The products were resolved by electrophoresis on a 1% w/v agarose gel in TAE buffer  and stained with ethidium bromide. Finally, DNA bands were quantified using a Gel Imaging Analysis System BINTA 2020D and the GelPro32 software . The primers were synthesized by Beijing AuGCT Biotechnology Co., Ltd, and sequencing of PCR products was performed by Beijing Tianyi Huiyuan Life Science & Technology, Inc. The following primers were used to detect the genomic DNA and mRNA:5′-TggTTCTTgggTTCTCCCgAAgCCT-3′,BR-L: 5′-AggTACCAATgTgTggCAgTCCA-3′,BR-R: 5′- AAATCCTCCCAAgCTgCTgCACg-3′,OV-L: 5′- AgTgCTgggCTTTCCCTCACTCA-3′.OV-R: http://www.compbio.net.cn/editfunc), a web server for predicting potential effects of RNA substitution editing, was used to predict the functional effects of the tissue-specific RNA editing sites. EditFunc can predict the effects of the RNA editing sites at the transcriptional level, including changes in canonical splice site sequences, exonic splicing enhancers, exonic splicing silencers, Piwi-interacting RNAs (piRNAs) and miRNAs compared with their targets. It can also predict the effects of RNA editing sites at the translational level, including alterations in the initiation codon, termination codon, amino acid residues, physicochemical properties, glycosylatioin sites, phosphorylation sites, propeptide cleavage sites and signal peptide domains.EditFunc , netOglyc Six EditFunc prediction options for propeptide cleavage site, signal peptide, N-linked glycosylatioin, O-linked glycosylatioin, C-linked glycosylatioin and Phosphorylation were used to first execute external programs Prop Table S1(DOC)Click here for additional data file.Table S2(DOC)Click here for additional data file.


Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor  Caenorhabditis elegans involves coordinated changes in expression of about a thousand genes. In order to find upstream regulators responsible for these aging genes, we used a genomics approach to screen for transcription factors that bind to them. We focused on one such regulator, unc-62/Homothorax, that encodes a co-factor for a Hox transcription factor. Although essential for development, expression of unc-62 in adults limits lifespan. We find that unc-62 is a transcriptional activator of yolk protein genes, which are synthesized in the intestine and encode essential nutrients for progeny but accumulate to toxic levels with age. Additionally, analysis of unc-62 knockdown indicates that when vitellogenin transcription is decreased, transcription of intestinal genes with somatic functions increases. Thus, activation of yolk protein gene expression by unc-62 is both a burden on maternal health and a necessary resource for embryonic growth. Surprisingly, we also found that the intestine of old worms has novel expression of non-intestinal proteins that are toxic when expressed in the intestine and are activated by unc-62 in old age. Our work on transcriptional regulation of intrinsic gene expression differences during normal aging has revealed insights into the mechanisms that limit lifespan.The normal aging process in  Caenorhabditis elegans, this includes loss of mobility and muscle cellular organization, decreased pharyngeal pumping, ectopic neuronal branching, deterioration of synapse function, and degeneration of the intestine The normal aging process involves the deterioration of a variety of tissues. In C. elegans have identified over a thousand transcripts that significantly increase or decrease in expression with age, covering a wide array of tissue-types and functions Genome-wide microarray studies of aging in et al. identified a hypodermal developmental circuit involving GATA transcription factor genes elt-3, elt-5, and elt-6 as an example of a developmental circuit affecting lifespan elt-3 declined with age independently of tested stress and damage signals, and increased activity of elt-3 led to increased lifespan. These results suggest that mis-regulation of developmental pathways during aging, including the elt-3 pathway, may be an intrinsic property limiting lifespan of the organism In an effort to link gene expression changes during aging to a regulator that mediates longevity, Budovskaya C. elegans (E.V.N. and S.K.K. unpublished data). The most promising transcription factors identified by this approach include not only regulators involved in stress response pathways, but also developmental regulators. One such factor, UNC-62, is the C. elegans ortholog of Hox co-factor Homothorax (Hth/Meis). UNC-62 plays essential roles in development of the nervous system, hypodermis, and vulva unc-62 beginning in adulthood extends lifespan by ∼40% Here we use a novel genomics approach based upon screening large-scale transcription factor target datasets generated by the modENCODE project to identify potential transcriptional regulators of age-dependent expression in C. elegans modENCODE consortium q-value<10−5) unpublished data; unpublished data; In order to uncover potential regulators of aging, we identified transcription factors with targets that are enriched for age-regulated genes. To identify targets bound by each transcription factor, we queried the data from 98 ChIP-seq datasets covering 57 transcription factors  generated by the p<10−5, we identified 9 transcription factors as potential aging regulators (E.V.N. and S.K.K. unpublished data). These include transcription factors that have been previously associated with aging and longevity, such as oxidative damage response factor skn-1elt-3Each set of factor-specific targets was then independently compared to a set of 1106 age-dependent genes obtained from DNA microarray experiments unc-62, has been shown to modulate lifespan C. elegans ortholog of Drosophila Homothorax and mammalian Meis, which are co-factors for HOX transcriptional regulators unc-62 activity yields phenotypes in maturation of the vulva, hypodermis, and the nervous system unc-62 activity by RNAi extends lifespan by ∼30–40% (−15). In thisunc-62 in specifying lifespan, we first set out to define the tissues in which it is expressed. Previous experiments using a 2.9 kb proximal promoter for unc-62 identified expression in vulval precursor cells, neurons, and some hypodermal cells unc-62 is expressed in the intestine unc-62 further, we made use of a GFP reporter for unc-62 in its full genomic context. This strain was generated by creating a modified fosmid with GFP inserted at the C-terminus of UNC-62, and includes regulatory elements contained within introns as well as distal promoter elements (To investigate the role of elements . Biolistle (s472).Using this translational fusion, we observed strong UNC-62:GFP expression in a variety of tissues in the hermaphrodite, including the vulval precursor cells, the ventral cord motorneurons and other neurons, the hypodermis, and the intestine . Expressunc-62 produces isoforms that differ in usage of the seventh exon (7a and 7b), which encodes the N-terminal region of the UNC-62 TALE homeodomain Alternative RNA splicing of unc-62 isoforms change with age in the intestine. We measured expression of the intestinal unc-62(7a) isoform during normal aging, and observed that it decreases in the intestine by day 8 of adulthood (unc-62(7a) mRNA levels between day 2 and day 8 of adulthood when measured by qPCR (unc-62(7b) was not observed in the intestine at any age. Thus, the decrease in expression of intestinal unc-62(7a) with age is not caused by changes in alternative splicing, but rather by a decrease in the level of transcription.We next determined whether expression of the  p<10−10). A simil by qPCR . In contunc-62 is expressed in a variety of tissues, we wanted to identify the tissues in which unc-62 functions to affect lifespan of the entire organism. To do this, we performed RNAi knockdown of unc-62 in strains that generate an RNAi response only in specific tissues. These strains contain a mutation in the RNAi pathway (rde-1) that leads to inactivation of the RNAi response, as well as a transgene expressing rde-1 under a tissue-specific promoter to restore the RNAi response only in that tissue  isoform.As t tissue  [24], . In order to better visualize intestinal expression, this strain was crossed with the glo-4(ok623) strain that lacks gut autofluorescence, and Is;glo-4(ok623) worms were isolated in the F2 generation (strain SD1897).To generate the ineering  [68]. Thunc-62(7a) and unc-62(7b), a single base in either exon 7a or 7b was mutated to generate an in-frame stop codon (Is;unc-119(ed3) (strain OP601) and Is;unc-119(ed3) (strain OP602) worms. These strains were similarly crossed to glo-4(ok623) worms to generate Is;glo-4(ok623) (strain SD1890) and Is was crossed into unc-62(s472) animals, and unc-62(s472); Is worms were isolated in the F2 generation using a PCR screening approach, yielding healthy strain SD1880. To further validate wild-type function, the full unc-62:GFP as well as the isoform-specific unc-62(7b):GFP fusions were crossed into unc-62(e644) worms containing a loss of function mutation in exon 7b, yielding (strain SD1887) and unc-62(7b):GFP;unc-62(e644)(strain SD1879) that were isolated by PCR screening of the F2 generation . Both of these rescued strains have viable phenotypes.To determine whether the full-length translational fusion was functional, unc-62: intestine-specific RNAi strain OLB11, hypodermal-specific RNAi strain NR222, muscle-specific RNAi strain NR350, neuronal- and vulval precursor cell-specific RNAi strain NK742, and uterine-specific RNAi strain NK640. To validate tissue-specific knockdown for intestine-specific and hypodermal-specific strains, these strains were crossed with the unc-62:GFP reporter, yielding SD1954 ]; rde-1(ne219); Is) and SD1955 ]; rde-1(ne219); Is). These worms were then placed on control or GFP RNAi as L1 larvae, and imaged as L4 larvae. To assay the effect of unc-62 RNAi on daf-16 activity, we used a previously-described reporter that expresses cytoplasmic GFP from the sod-3 promoter Five tissue-specific RNAi strains from previous publications were used to test the tissue of action for C. elegans techniques as well as lifespan experiments were performed as previously described General unc-62, these exons (as well as ∼50 bp of flanking introns) were PCR amplified and inserted into the L4440 vector at the EcoRV restriction site, and transformed into HT115(de3) E. coli. RNAi knockdown experiments in adults (including lifespan experiments) were performed on NGM plates supplemented with 30 uM FUDR, 100 ug/mL Ampicillin, and 2 mM IPTG to induce dsRNA expression.RNAi clones used were obtained from the Ahringer RNAi library Dataset S1unc-62 RNAi and control RNA-seq experiments. Rank products analysis is included for all 7333 genes with 10 or more reads observed in all three control, or all three unc-62 RNAi, RNA-seq replicates. Each gene was ranked in each biological replicate experiment according to the observed fold-change in unc-62 RNAi as compared to control. The product of ranks across three biological replicates was used to determine the false-positive rate for observing such reproducible fold-change .Rank products analysis of (ZIP)Click here for additional data file.Figure S1q-value<10−5) in 98 ChIP-seq datasets generated by the modENCODE consortium, with UNC-62 young adult binding sites in red and all other binding sites in black. (A) An UNC-62 binding site in young adults proximal to vit-2 is factor-specific; the binding site is not significantly enriched in other ChIP-seq experiments. We defined factor-specific targets as those that are significantly enriched in nine or less transcription factors profiled. (B) An UNC-62 young adult binding site in the rpl-15 promoter is not factor-specific, as many other transcription factors also bind to that region.Examples of factor-specific and non-specific UNC-62 targets. (A–B) Examples of factor-specific and redundant UNC-62 binding sites are shown in genome browser snapshots. Genes are indicated in blue at the top, with exons (boxes) and introns (lines) indicated. Boxes below indicate all regions significantly enriched ((TIF)Click here for additional data file.Figure S2p<10−4 by Student's t-test). (B) unc-62(7a) mRNA decreases by qRT-PCR. RNA from ∼75 day 2 and day 8 adult worms (grown on empty vector RNAi) was purified, and qPCR was performed using primers specific to exon 7a of unc-62. Each bar indicates average expression from a biological replicate, and error bars indicate the standard deviation among two qPCR technical replicates. For each, expression was first normalized to an htz-1 control, and then calculated relative to unc-62(7a) at day 2 of adulthood.UNC-62(7a) expression decreases with age. (A) UNC-62(7a):GFP fluorescence was quantified in the first pair of intestinal nuclei at various ages. Bar height indicates average fluorescence observed per intestinal nuclei for 32–44 worms quantified at each age, with error bars indicating standard error of the mean. We validated these results in an independent sample (right). In all three cases, day 1 adults had significantly higher expression than day 3, 8, or 12 (each (TIF)Click here for additional data file.Figure S3unc-62:GFP reporter. Worms were then placed on control and GFP RNAi as L1 larvae, and imaged as L4 larvae. (A) GFP RNAi in intestine-specific RNAi strain SD1855 (generated from strain OLB11) does not affect hypodermal (hyp.) or neuronal/ventral nerve cord (vnc) expression of unc-62:GFP, but decreases unc-62:GFP expression in the intestine (int). Although most intestinal cells showed complete loss of unc-62:GFP expression, some nuclei still had visible unc-62:GFP expression (as indicated). (B) GFP RNAi in hypodermal-specific RNAi strain SD1854 (generated from strain NR222) does not affect intestinal or neuronal/ventral nerve cord expression of unc-62:GFP. However, hypodermal expression of unc-62:GFP was no longer visible.Tissue-specific RNAi fluorescence validation. To verify that the intestine- and hypodermal-specific RNAi strains function as intended, we crossed in the (TIF)Click here for additional data file.Figure S4unc-62. (A) Wild-type (N2) worms were grown on control bacteria, and then shifted to unc-62 at various days of adulthood to determine the time of effect of unc-62 knockdown. unc-62 RNAi significantly increased lifespan when begun at days 1, 3, or 5 of adulthood . RNAi of unc-62 beginning at days 7 or 9 of adulthood did not significantly extend lifespan (p = 0.15 and 0.63 respectively). As the rate of feeding declines significantly with age, we do not know whether the lack of effect observed at day seven indicates that unc-62 no longer limits lifespan or simply reflects the weakened effect of RNAi by feeding. (B) Lifespan of knockdown of unc-62, ins-7, and combined RNAi targeting both unc-62 and ins-7 was performed in a strain expressing rde-1 from the elt-2 intestinal promoter (strain OLB11). Intestine-specific ins-7 knockdown extends lifespan 8.3% (p<10−5 by log-rank test). Combined RNAi targeting both unc-62 and ins-7 showed a 6.1% increase over unc-62 alone (p = 0.0347), suggesting that unc-62 RNAi by itself may not completely silence ins-7 expression in the intestine. (C) RNAi of unc-62 in germ-line mutant glp-1(e2141) worms significantly extends lifespan . To remove the germ-line, glp-1(e2141) worms were grown at the restrictive temperature (25°C) until the first day of adulthood, and then shifted to 20°C.Lifespan analysis of (TIF)Click here for additional data file.Figure S5unc-62 RNAi, we developed an analysis method based on the Rank Products method previously described for microarray analysis unc-62 RNAi or control bacteria, isolated mRNA and generated RNA-seq libraries, and sequenced these libraries on the Illumina HiSeq platform. For analysis, we discarded genes that were not covered by at least 10 sequencing reads in either controls or unc-62 knockdown across all three replicates (red arrows). (B) For each gene in each replicated experiment, the fold-change was calculated between experimental (unc-62 RNAi) and control. These fold-changes were then ranked for each replicate. (C–D) A rank product (RP) was then calculated for each gene as the product of the fold-change ranks in each replicate, and converted to a probability. The ranks were then permuted 10,000 times to generate the expected number  of genes observed with a probability less than or equal to the observed RP. This E-value could be converted to a false-positive rate by dividing by the number of genes actually observed to have such a RP probability. (C) Genes were ranked by increasing fold-change upon unc-62 RNAi (starting with the most down-regulated gene) to calculate a false-positive rate for decreased expression upon unc-62 RNAi. (D) Genes were ranked by decreasing fold-change upon unc-62 RNAi (starting with the most up-regulated gene) to calculate a false-positive rate for increase expression upon unc-62 RNAi.Rank products method for identifying differential expression in biologically replicated RNA-seq experiments. In order to identify genes that were consistently increased or decreased in expression upon (TIF)Click here for additional data file.Figure S6vit-2 and vit-6 promoters in young adult worms. OP600 (unc-62:GFP) worms were grown on peptone plates until adulthood, at which point they were switched to NGM plates containing 30 µM FUDR until day 4 of adulthood. ChIP was performed using the protocol from the modENCODE consortium vit-2 and vit-6 promoter regions as well as three regions bound by other transcription factors but not UNC-62 . For each, fold-enrichment was calculated by comparing first to a non-immunoprecipitated input sample, and then to the first control promoter region (B0222.8). Bars indicate mean, and error bars indicate standard deviation from technical triplicate qPCR measurements.ChIP-qPCR validates UNC-62 association at (TIF)Click here for additional data file.Figure S7De novo motif searches identify an ATTGACA motif among UNC-62 adult binding sites. (A) Two of the three motifs identified in a de novo motif search of UNC-62 young adult binding sites with RSAT p-values were determined by Fisher's Exact test.(TIF)Click here for additional data file.Figure S8unc-62 RNAi. Histograms indicate the percent of these gene sets with indicated fold-changes in expression (shown in 0.1 (log2) increments) in RNA-seq of adult worms exposed to unc-62 RNAi. Datasets were obtained from previous publications for genes with enriched expression in (A) muscle cells unc-62 RNAi , whereas muscle- and neuronal-enriched genes are not (p>0.01 by Kolmogorov-Smirnov test).Intestine-enriched genes are uniquely shifted towards higher expression upon (TIF)Click here for additional data file.Figure S9let-70 and tbb-2), two intestine-specific positive controls (ges-1 and vit-6), and four non-intestinal genes: pharynx-specific myo-2, germline-specific pie-1, neuronal unc-119, and hypodermis and gonad-expressed lin-26. RNA was isolated from micro-dissected intestines as described in Methods, and reverse transcribed into cDNA for quantification by qPCR. Bars indicate ΔΔCt values are calculated as - , using reference M7.1. All Ct values are averaged from triplicate technical replicates. Error bars indicate standard deviation of triplicate technical replicates. * indicates genes that were undetectable in two of three technical replicates; for these, the expression in the single amplified replicate was used to estimate expression. ** indicates genes that did not amplify from the indicated sample; for these experiments, an upper bound estimate of expression of 40 cycles was used.Dissected intestines are enriched for intestine-specific transcripts and depleted for non-intestinal transcripts. Transcripts include two ubiquitously expressed reference genes ((TIF)Click here for additional data file.Figure S10tsp-1 and tsp-2 are activated by UNC-62 in old adult intestines. An UNC-62 young adult binding site (indicated by the red box) located just upstream of tsp-2 was initially associated with tsp-1, as the point of maximal read density was located within the tsp-1 transcript sequence . Error bars indicate standard deviation from triplicate technical replicates.Both sequence . We perf(TIF)Click here for additional data file.Table S1unc-62 RNAi. “Post-quality filtering” indicates reads that did not contain a stretch of ten or more consecutive A or T nucleotides (which were discarded as likely artifacts from the reverse transcription oligonucleotide). Reads were mapped to the WS215 genome and annotated transcripts with Bowtie (see Methods), and were then discarded if they mapped with an equal number of mismatches to multiple positions in the genome. To simplify downstream analyses, at the final step only reads mapping to the sense strand of annotated transcripts were used. “Vitellogenin transcripts” indicates the number of reads in each experiment that mapped to the six vitellogenin loci .RNA-seq experiment summary information. Values indicate number of reads meeting the stated criteria observed from RNA-seq experiments performed on three biological replicates of day four adult worms grown either on empty vector control, or (XLSX)Click here for additional data file.Table S2p-values were calculated by log-rank test in Oasis E. coli until the first day of adulthood and then shifted to bacteria expressing the indicated dsRNA. For tissue-specific RNAi experiments, the Tissue column indicates the tissue in which the RNAi pathway is active. For time of action experiments, worms were shifted to HT115 (empty vector) bacteria on the first day of adulthood, and then shifted to unc-62 RNAi bacteria at the indicated day for the remainder of lifespan.Data for lifespan experiments. Each row in the table describes data from a lifespan experiment described in the text, grouped by experiments performed at the same time. (XLSX)Click here for additional data file.Table S3unc-62 RNAi. Each row indicates a gene associated with an UNC-62 binding site in day four young adults that was identified to be significantly altered upon unc-62 RNAi in the RNA-seq experiment. Fold-change is shown as the average fold-change from the three biological replicates . Distance to TSS indicates the distance from the annotated transcription start site to the point of maximal read density within the ChIP-seq peak. For genes with multiple isoforms, the shortest distance is reported.Genes bound by UNC-62 and differentially regulated upon (XLSX)Click here for additional data file.


Canine coronavirus (CCoV) is usually responsible for mild, self-limiting infections restricted to the enteric tract. We report an outbreak of fatal disease in puppies caused by a pathogenic variant of CCoV that was isolated from organs with severe lesions. The same symptoms were observed 3–4 days later in 2 other miniature pinschers (45 days of age) and 1 Pekinese (56 days of age). Necropsy of the dogs showed hemorrhagic enteritis, abundant serosanguineous fluid in the abdominal cavity, and severe lesions in the parenchymatous organs. The lungs had multiple, patchy, red areas of consolidation. Livers were yellow-brown and congested, with hemorrhages on their surfaces, and spleens were enlarged with subcapsular hemorrhages. Variable gross changes in other organs included multifocal hemorrhagic renal cortical infarcts and petechial hemorrhages on lymph node surfaces.6 RNA copies/μL of template), spleen (median 4.46 × 106 RNA copies/μL of template), liver (median 9.02 × 104 RNA copies/μL of template), kidney (median 7.54 × 104 RNA copies/μL of template), and brain (median 5.23 × 103 RNA copies/μL of template). Virus-induced cytopathic effect was observed in A-72 cells, and CCoV type II strain (CB/05) was isolated from all tissues examined except brain tissue. Immunohistochemical analysis with a CCoV-specific monoclonal antibody detected CCoV antigen in the organs with gross lesions that were examined   assays (h nodes) .,The sequence of the 3´ end of the genome (8.8 kb) of the pantropic CCoV strain was determined by RT-PCR amplification and sequencing of overlapping fragments. The S, envelope, and membrane proteins and nucleoprotein showed a high degree of amino acid identity with the cognate open reading frame (ORF) of CCoV type II. The S protein of strain CB/05 had the highest identity to FCoV type II strain 79-1683 . Compari5.50/50 μL on A-72 cells and 1.18 × 107 RNA copies/μL of template by real-time RT-PCR. The virus was reisolated from the experimentally infected dogs. Severe clinical symptoms characterized by pyrexia (temperature 39.8°C–40.1°C), anorexia, depression, vomiting, diarrhea, and leukopenia were observed that persisted 8–10 days. Despite the severe symptoms, the dogs slowly recovered from their illness.To confirm the pathogenic potential of strain CB/05, we experimentally infected two 6-month-old dogs . Two milliliters of cryolysate of a lung-derived first-passage virus in A-72 cells were administered intranasally to the dogs. The cell cryolysate tested negative for other common canine pathogens and had a 50% tissue culture infectious dose of 10–,Point mutations or deletions in the S protein and NSPs have been associated with changes in tropism and virulence of coronaviruses  likely resulted in the disease being nonfatal. Accordingly, the appearance of pathogenic CCoV variants should always be regarded as a potential threat to domestic dogs and considered when unexplainable fatal disease outbreaks occur in puppies.Epidemiologic studies are required to determine whether the pantropic CCoV strain is a new coronavirus variant emerging in canine populations or a widespread infectious agent of dogs that usually goes undetected. Vaccination trials could also help determine whether the CCoV vaccines currently available are effective against the highly virulent CCoV strain.The 2002–2003 SARS epidemic has demonstrated that the study of animal coronaviruses is paramount to understanding the ecology and evolution of human coronaviruses. The coronaviruses of carnivores provide a paradigmatic model of how coronaviruses cross the species barriers, adapt to new host species, and change their pathogenicity.


Paramphistomum cervi (Digenea: Paramphistomidae). This genome  is slightly larger than that of Clonorchis sinensis , but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke,  Paramphistomum cervi is perhaps the most widespread species, being reported from many parts of Eurasia P. cervi has been subjected to immuno-blotting using sera from bovines infected with P. cervi. This method, however, has not been widely adopted Paramphistomosis, due to paramphistomes (Trematoda: Digenea: Paramphistomidae), has recently emerged as a major cause of productivity loss in ruminants. Adult worms often inhabit the rumen and reticulum of cattle, water buffaloes, sheep and goats. Their presence in these sites may elicit few apparent signs or symptoms. However, acute parasitic gastroenteritis causing high morbidity and mortality may occur as large numbers of immature paramphistomes migrate through the intestine towards the rumen and reticulum. Severity of disease is greatest in young animals Rapid development of molecular biology techniques, especially the polymerase chain reaction (PCR), may provide valuable supplementary tools for the differential identification of digenean infection to overcome limitations of current morphological-diagnostic methods. Due to their high nucleotide substitution rates, partial or complete mitochondrial (mt) genomes of parasitic flatworms have become very popular markers for detecting their presence in animals and for investigating their phylogenetic relationships at different levels P. cervi is a major limitation for the development of molecular diagnostic techniques, for analyses of population and genetic variation within this species, and for phylogenetic studies of the Digenea in general.The lack of knowledge of mt genomics for P. cervi, which was collected from Qinghai Province, China. Phylogenetic analyses were performed using concatenated mt sequences of 12 protein-coding genes of digenean species available in GenBank to date. The new mt genome sequence may provide useful information on both genomics and the evolution of Paramphistomidae, because there are no complete (or nearly complete) mtDNA sequences available from any member of this family.In our present study, we determined the complete mt nucleotide sequence of P. cervi adults were collected was being processed at a local abattoir in Dari County, Qinghai Province, as part of the normal work of the abattoir.The yak from which P. cervi  were collected from the rumen of a naturally infected yak in Dari County, Qinghai Province of China. The flukes were washed extensively in physiological saline and identified to species in the Key Laboratory of Veterinary Parasitology, Gansu Province based on morphological characters (collection accession number: 20110101).Adult 2O, followed by RNase treatment step. The treated DNA sample was stored at −20°C until use.Total genomic DNA was extracted from one parasite using a Qiagen Blood and Tissue Kit  according to the manufacturer's instructions and eluted into 100 μl HFasciola hepaticaClonorchis sinensisOpisthorchis felineusParagonimus westermani (GenBank Accession No. AF219379) . The cycling conditions used were 94°C for 5 min ; then 94°C for 1 min (denaturation), 50°C for 35 s , 72°C for 1–3 min (extension) for 30 cycles and a final extension at 72°C for 10 min. Each PCR reaction yielded a single band detected in a 1.0% (w/v) agarose gel stained with ethidium-bromide P. cervi was assembled using DNAStar software as a sequence editor Amplification, sequencing and assembly of mtDNA fragments was performed according to methods previously described F219379) . These shttp://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to find protein-coding gene sequences, which were subsequently used to search for homologous digenean sequences deposited in the GenBank™ by using tBLASTn. The rhabditophoran platyhelminth genetic code The ORF finder tool at NCBI (rrnL) and small (rrnS) subunit ribosomal RNA genes were identified by comparison with the mt rRNA genes of F. hepatica, C. sinensis, O. felineus, P. westermani and other flatworms Putative tRNA genes were identified using the program tRNAscan-SE http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=tgencodes#SG9), the concatenated amino acid sequences were aligned using Clustal P. cervi and 11 other digenean species (Echinococcus granulosus (NC_008075) was used as an outgroup.DNA sequences of the 12 protein-coding genes were concatenated and imported into BioEdit  species . The mt In every case two runs, each of four chains, were specified. For the nucleotide alignment, the GTR+I+G model was as described previously For the amino-acid alignment, MrBayes was allowed to determine the most appropriate model (“prset aamodelpr = mixed”), 2,000,000 generations were run and trees sampled every 500. The first 25% of trees were omitted as burnin.P. westermani (AF219379) is around 21 kb Schistosoma japonicum (AF215860) and S. mansoni (AF216698), it is 16.5–24 kb S. spindale (NC_008067) has a total length of 16,901 bp and Trichobilharzia regenti (NC_009680), also a member of the Schistosomatidae, has a very short non-coding region and a total length of 14,838 bp S. turkestanicum is 14,755 bp Lengths cited for some “complete” digenean mt genomes in GenBank are incorrect. They are instead the lengths of the coding portions. For these species, amplification and sequencing of non-coding regions proved impossible because of the presence of numerous repeats and other features. Complete mt genome lengths for these were inferred from Southern blotting experiments P. cervi  is 14,014 bp in length, within the range of typical sizes for metazoan mt genomes (14–18 kb). The mt genome of P. cervi is larger than that of C. sinensis , but smaller than those of other digenean species available in GenBank™ to date , 22 transfer RNA genes and 2 ribosomal RNA genes (rrnL and rrnS)   and 3. Anad4L and the 5′ end of nad4 was noted in P. cervi, similar to that of other digeneans.A 40 bp overlap between the 3′ end of P. cervi mt genome. The nucleotide composition in P. cervi was biased toward G and T, which is similar to that of the digeneans F. hepatica, O. felineus, C. sinensis, P. westermani and the outgroup cestode, E. granulosus, but is slightly different from S. turkestanicum, S. japonicum and other schistosomes, which are biased toward A and T. In the protein-coding genes of P. cervi, strong bias against the usage of C  and strong bias in favor of the usage of T  were observed. The frequency of usage for G  was higher than that for A   .F. hepaticaO. felineus and C. sinensisS. turkestanicumS. spindale and S. haematobiumP. cervi. It is interesting that the stop codon TAG was used for all the mt protein-coding genes of P. cervi. This is unusual, because another termination codon, TAA, is often observed in other digeneans.The most common inferred start codon for mt protein-encoding genes of digenean species is ATG, followed by GTG (e.g. (AGN) and tRNA-Cys, all tRNA genes appear to exhibit the standard cloverleaf structure. The predicted secondary structure of the serine tRNA(AGN) contains the TΨC arm but lacks the DHU arm  O. felineusP. cervi relative to the situation in F. hepatica, P. westermani and the opisthorchiids , suggesting that this change in tRNA gene position could provide an important phylogenetic signal Except for tRNA-Ser1rrnL  and rrnS  genes of P. cervi were identified by sequence comparison with those of F. hepatica, O. felineus, and Schistosoma spp. These two genes are separated by tRNA-Cys. The sizes of the rrnL and rrnS genes were 986 and 749 bp, respectively, and their A+T content was 63.59% and 60.75%, respectively, which are the lowest among the digeneans studied to date  and in P. westermani . In other species, notably in the genera Opisthorchis and Clonorchis, the non-coding regions lack strong structures, such as large repeats. There does not seem to be a strong phylogenetic element to length and structure of the LNR. In P. cervi, there is a short non-coding region (SNR) (58 nucleotides), lacking any notable features and located between cytb and nad4L. A long non-coding region (LNR) (499 nucleotides), is observed between tRNA-Glu and cox3  that are often separated by one or more tRNA genes. A common feature of LNRs is the presence of long repeats. Such features are found in and cox3  and 4. S the SNR  and 4. AP. cervi was sister to this clade (in 70% of trees). In 30% of trees, P. cervi was placed basal to the other plagiorchiids  as most other digeneans, consisting of 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes. Phylogenetic trees, based on sequences of protein-coding genes, could identify the two orders represented (Diplostomida and Plagiorchiida). For members of the Diplostomida, relationships are exactly as expected from other studies. For the five members of the Plagiorchiida, results are not as consistent, but our sampling of this clade is very sparse and additional sequences are needed. The complete mtDNA sequence of P. cervi will add the knowledge to digenean mitochondrial genomics. It will also provide an important resource for the studies of inter- and intra-specific variation of the Paramphistomidae and a resource for comparative mitochondrial genomics and systematic studies of digeneans.In conclusion, the present study determined the complete mt genome sequence of


RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods.Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices.This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library  replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates. RNA sequencing (RNA-Seq) allows an entire transcriptome to be surveyed at single-base resolution whilst concurrently profiling gene expression levels on a genome scale. RNA-SeqArguably, the most popular use of RNA-Seq is profiling of gene expression or transcript abundance between samples or differential expression (DE). The efficiency, resolution and cost advantages of using RNA-Seq as a tool for profiling DE has prompted many biologists to abandon microarrays in favour of RNA-Seq,8.Despite the advantages of using RNA-Seq for DE analysis, there are several sources of sequencing bias and systematic noise that need to be considered when using this approach. Clearly, RNA-Seq analysis is vulnerable to the general biases and errors inherent in the next-generation sequencing (NGS) technology upon which it is based. These errors and biases include: sequencing errors , biases in sequence quality, nucleotide composition and error rates relative to the base position in the read,10, variRecently, there have been several investigations-15 into Despite the known biases, RNA-Seq continues to be widely and successfully used to profile relative transcript abundances across samples to identify differentially expressed transcripts. The proGood experimental design and appropriate analysis is integral to maximising the power of any NGS study. With regard to RNA-Seq, important experimental design decisions include the choice of sequencing depth and number of technical and/or biological replicates to use. For researchers with a fixed budget, often a critical design question is whether to increase the sequencing depth at the cost of reduced sample numbers or to increase the sample size with limited sequencing depth for each sample.Sequencing depth is usually referenced to be the expected mean coverage at all loci over the target sequence(s), in the case of RNA-seq experiments assuming all transcripts having similar levels of expression. Without the benefit of extensive previous RNA-Seq studies, it is difficult in most cases to estimate prior to data generation the optimal sequencing depth or amount of sequencing data required to adequately power the detection of DE in the transcriptome of interest. Pragmatically, RNA-seq sequencing depth is typically chosen based on an estimation of total transcriptome length (bases) and the expected dynamic range of transcript abundances. Given the dynamic nature of the transcriptome, the suitability of these estimates could vary substantially across organisms, tissues, time points and biological contexts.Wang et al. found a Replication is vital for robust statistical inference of DE. In the context of RNA sequencing, multiple nested levels of technical replication exist depending upon whether it is the sequence data generation, library preparation or RNA extraction technical processes that are being replicated from the same biological sample. Several published studies have incorporated technical replicates into their RNA-Seq experimental designs-25. The n is increased from n = 2 to n = 5 if the package is performing correctly.To test the performance of each package under the null hypothesis, we simulated sets of n and all packages. This is a known artifact of calculating p-values for discrete random variables using the method described in. A: TPR padj ≤ 0.01. B: FPR padj ≤ 0.01. The solid grey line (“multiplex line”) connecting the TPR values of n biological replicates atn are used despite the loss power due to the sequencing depth reduction required by the multiplexing of lanes. This trend remains true even for the n = 32 and n = 96 cases.Click here for fileFigure S4. Venn-diagram showing the TP and FP calls made by DESeq  and edgeR  and how they overlap between each other and the total pool of transcripts designated as truly DE . A: the Venn-diagram for the case in which the number of biological replicates is n = 12 and depth is 100%. This combination of n and depth is somewhat unrealistic as the cost of 24 lanes of sequencing would be almost prohibitive; however, it shows a ‘best case scenario’ situation in which 2870 of the total 5689 truly DE transcripts were detected by the union of DESeq and edgeR. Of these 2870 TP detections, most of them (2360) were detected by both algorithms – hence either algorithm would have sufficed. B: the Venn-diagram for n = 4 and depth is 25%. This more realistic experimental design choice of n and depth shows the value of using both algorithms; only 913 out of the total 5697 truly DE transcripts were detected by both algorithms, only two thirds of them (591) were detected by both algorithms. These contrasting scenarios show that the use of both algorithms aids to further constrain the list of viable DE candidates in a fast and cheap manner.Click here for fileNegative binomial modelc.Click here for fileFigure S3. Smallest fold-change required for a transcript to be called DE (padj ≤ 0.01) as a function of n biological replicates (using DESeq). The more replicates available; the smaller the fold-change required for a transcript to be called DE by DESeq or edgeR.Click here for fileFigure S1. Maximum likelihood estimates of the NB meanα = 1.700, c = 0.364.Click here for file


To the Editor: Hepatitis E virus (HEV) is a positive-stranded, non-enveloped RNA virus of the family Hepeviridae that is considered to be the main causative agent of enterically transmitted acute hepatitis  in abattoirs in Douala and Yaoundé, Cameroon, and in slaughter slaps (areas) in Bamenda, Cameroon. Pigs were mainly of the local breed. In addition, pigs originating from extensive cross-breeding  were sampled. Liver samples were collected during post-mortem inspection.,Viral RNA was extracted from liver samples by using the RTP DNA/RNA Virus Mini Kit II  according to the manufacturer’s instructions. Extracted RNA was analyzed for HEV RNA by using 2 nested reverse transcription PCRs (RT-PCRs) specific for open reading frame 1 (OFR 1) and ORF 2 of HEV  and 1 sample from a male pig in Bamenda (1/39). All 167 samples from Douala were negative for HEV RNA. The sample from Bamenda showed a positive result for the nested RT-PCR specific for HEV ORF 1. Genetic distances calculated with partial nucleotide sequences of ORF 1 (280 nt) and ORF 2 (373 nt) between strain Yaounde56 and the most closely related HEV genotype 3 strains from Japan  and Mongolia  were 90% and 91%, respectively.At the amino acid level, the partial RNA-dependent RNA polymerase sequence (ORF 1) and the partial capsid protein sequence (ORF 2) of strain Yaounde56 were identical to those of HEV genotype 3 strains HEV/Gt3/HSD40/2009 (GenBank accession no. AFO71833) from Germany and swJ12–1 (GenBank accession no. BAC66273) from Japan. Thus, all mutations were silent.In agreement with distance analysis, phylogenetic reconstruction using partial nucleotide sequences of ORF 2 (278 nt) showed a close relationship of strains Yaounde56 and Yaounde94 with HEV genotype 3 strains . HoweverBecause the pig production cycle is shorter than that for cattle, pig production is a major economic activity in Cameroon. Most pigs in Cameroon are local raised, and extensive cross-breeding is used. The infection rate of pigs with HEV genotype 3 strains from Cameroon is lower than that of pigs from Europe. Thus, HEV genotype 3 seems to have an extensive distribution that includes Africa. Future studies should investigate how HEV genotype 3 strains contribute to sporadic HEV cases in Cameroon.