Rice Global Transcriptome Analyses
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Project description:Lysine crotonylation (Kcr) is a newly discovered post-translational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice (Oryza sativa L. japonica) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome, and might mark other cis-regulatory DNA elements which are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants.
2018-09-20 | PXD010376 | Pride
Project description:We have performed a Proteogenomics meta-analysis of data sets deposited in ProteomeXchange: PXD000265, PXD000313, PXD000923, PXD001030, PXD001058, PXD002291, PXD002739, PXD002740 and PXD003156 and using 29 RNA-Seq data sets on rice (Oryza sativa). We created a search database comprising translated reads that had been mapped onto the rice genome, as well as officially annotated rice proteins sequences. The RNA Seq database was pre-processed to identify “novel transcripts” for those not mapping fully to an existing exon, and “novel junctions” for those reads mapped with a gap, implying a potential novel splice site that was not annotated in the official gene set. Confidentially identified “novel peptides” i.e. those mapping to a novel junction or novel transcript were post-processed to ensure that there were no other better explanations for the corresponding spectra e.g. peptide from a canonical gene with a modification or amino acid substitution. Data were exported from the pipeline in PSI mzIdentML 1.2 format, containing chromosomal coordinates, and further converted to PSI proBed format for genome visualisation. Novel peptides were searched against other plant databases using BLAST to see if they had predicted in genes from other species. A total of 1584 novel peptides were identified, mapping to ~700 genomic loci in which either new genes have been predicted (~100) or updates to existing gene models have been predicted (~600).
2018-10-30 | PXD008960 | Pride
Project description:Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.
2019-09-24 | GSE137883 | GEO
Project description:Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or a few conditions. We applied direct high-throughput sequencing of cDNAs, complemented with different expression data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including exponential proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at unprecedented resolution. Unlike hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not actively transcribed or rapidly degraded. The combined sequencing and strand-specific array data provided rich information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus refining the existing genome annotation. Sequence trans-reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes. Splicing efficiency was largely coordinated with transcriptional efficiency, and hundreds of introns showed regulated splicing as a function of cellular proliferation or differentiation.
2008-05-18 | E-MTAB-18 | biostudies-arrayexpress
Project description:Recent data from several organisms indicate that the transcribed portions of genomes are larger and more complex than expected, and many functional properties of transcripts are not based on coding sequences but on regulatory sequences in untranslated regions or non-coding RNAs. Alternative start and polyadenylation sites and regulation of intron splicing add additional dimensions to the rich transcriptional output. This transcriptional complexity has been sampled mainly using hybridization-based methods under one or a few conditions. We applied direct high-throughput sequencing of cDNAs, complemented with different expression data from high-density tiling arrays, to globally sample transcripts of the fission yeast Schizosaccharomyces pombe, independently from available gene annotations. We interrogated transcriptomes under multiple conditions, including exponential proliferation, meiotic differentiation and environmental stress, and in RNA processing mutants, to reveal the dynamic plasticity of the transcriptional landscape as a function of environmental, developmental, and genetic factors. High-throughput sequencing proved to be a powerful and quantitative method to deeply sample transcriptomes at unprecedented resolution. Unlike hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect widespread transcription in >90% of the genome, including traces of RNAs that were not actively transcribed or rapidly degraded. The combined sequencing and strand-specific array data provided rich information on novel, mostly non-coding transcripts, untranslated regions and gene structures, thus refining the existing genome annotation. Sequence trans-reads spanning exon-exon or exon-intron junctions gave unique insight into a surprising variability in splicing efficiency across introns and genes. Splicing efficiency was largely coordinated with transcriptional efficiency, and hundreds of introns showed regulated splicing as a function of cellular proliferation or differentiation.
2008-12-10 | E-MTAB-5 | biostudies-arrayexpress
Project description:Single-cell isoform regulation is increasingly being studied. To get a full view of alternative isoform usage, from transcription start site and alternative splicing to transcription termination site, full-length sequences have to be studied. Here we use PacBio long read sequencing technology combined with unique molecular identities to get a full and accurate picture of alternative isoform usage of different stages in maturing oligodendrocyte single cells. We see that the majority of molecules in single-cells are separate isoforms, even after applying a conservative definition of what constitutes an isoform. Few isoforms are common between cells of the same cell-type but the common isoforms are higher expressed. We also see that exon junctions in coding regions are better regulated than exon junctions in non-coding regions and that genes often express more than one coding isoform.
2017-01-01 | GSE76026 | GEO
Project description:Signaling pathways are controlled by a vast array of post-translational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a novel key element of this pathway. The EJC binds the exon-exon junctions of mRNAs, and thus far, has been linked exclusively to post-splicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC.
2010-10-20 | GSE24012 | GEO
Project description:The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex which binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit RnpS1 in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where in the absence of RnpS1 the fourth intron of piwi is retained. Within this intron the polypyrimidine tract is disrupted by a transposon-adjacent A/T-rich sequence that confers dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. Small-RNA libraries from two control samples and four knockdowns in germline or somatic tissues of the Drosophila melanogaster ovary.
2014-08-14 | E-GEOD-59327 | biostudies-arrayexpress
Project description:This experiment uses iCLIP to identify the binding pattern of the spliceosomal protein PRPF8 on RNA. The data shows that PRPF8 binds strongly and specifically in the region 12 to 14nt upstream of 5' splice sites (5ss). Due to PRPF8's role in the formation of the catalytically active spliceosome, this data can be used as a readout of 5ss selection. Here, we performed iCLIP on HeLa cells treated with control or EIF4A3 siRNA, with 4 replicate samples per condition and eIF4A3 protein levels reduced ~50% in knockdown. We investigated the role of the exon junction complex (EJC) in suppressing 5ss that are reconstituted at the junction of two canonical exons (RS-5ss) - selection of these splice sites would result in recursive splicing of canonical exons. We plotted the crosslink sites of reads that span an exon-exon junction, seperating reads that span RS-5ss from those that do not. We found that reads that span an RS-5ss are enriched at the 12-14nt window associated with 5ss selection, while reads that span other exon-exon junctions are not enriched. This effect is magnified greatly by knockdown of eIF4A3. The results indicate that RS-5ss can be used by the spliceosome, but that this process is usually repressed by the EJC. This data is evidence of recursive splicing of canonical exons and the role of the EJC in repressing recursive splicing.
2018-10-14 | E-MTAB-7269 | biostudies-arrayexpress
Project description:Signaling pathways are controlled by a vast array of post-translational mechanisms. By contrast, little is known regarding the mechanisms that regulate the expression of their core components. We conducted an RNAi screen in Drosophila for factors modulating RAS/MAPK signaling and identified the Exon Junction Complex (EJC) as a novel key element of this pathway. The EJC binds the exon-exon junctions of mRNAs, and thus far, has been linked exclusively to post-splicing events. Here, we report that the EJC is required for proper splicing of mapk transcripts by a mechanism that apparently controls exon definition. Moreover, whole transcriptome and RT-PCR analyses of EJC-depleted cells revealed that the splicing of long intron-containing genes, which includes mapk, is sensitive to EJC activity. These results identify a role for the EJC in the splicing of a subset of transcripts and suggest that RAS/MAPK signaling depends on the regulation of MAPK levels by the EJC. Transcriptome sequencing (RNA-Seq) of Drosophila S2 cells to monitor the effect of EJC depletion on the cellular mRNA expression profile. Each treatment (dsRNA knockdown of MAGO (CG9401), dsRNA knockdown of eIF4AIII (CG7483)) was done in biological duplicate and each sample was sequenced separately on a quad slide on the SOLiD 3.0 platform. The reference samples were treated with a dsRNA targeted to GFP.
2010-10-19 | E-GEOD-24012 | biostudies-arrayexpress