Global co-transcriptional splicing in Arabidopsis and the correlation with splicing regulation in mature RNAs
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ABSTRACT:
PROVIDER: PRJNA520905 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8528220_1.fastq.gz | Fastqsanger.gz | |||
| SRR8528220_2.fastq.gz | Fastqsanger.gz | |||
| SRR8528230_1.fastq.gz | Fastqsanger.gz | |||
| SRR8528230_2.fastq.gz | Fastqsanger.gz | |||
| SRR8528221_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we showed that in Arabidopsis thaliana, nearly all introns undergo co-transcriptional splicing and this occurs with higher efficiency for introns in protein-coding genes than for noncoding RNAs. Co-transcriptional splicing efficiency correlates with density of 19 epigenetic modifications. Total intron number and intron position are two predominant sequence features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5′ or 3′ splice sites are less efficiently spliced. Furthermore, we found that trans-acting protein mutations lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Our results not only uncovered widespread co-transcriptional splicing in Arabidopsis, but also uncovered features that may affect or be affected by co-transcriptional splicing efficiency.
2019-11-25 | GSE126064 | GEO
Project description:RNA splicing and spliceosome assembly in eukaryotes occur mainly during transcription. However, co-transcriptional splicing has not yet been explored in plants. Here, we built transcriptomes of nascent chromatin RNAs in Arabidopsis thaliana and showed that nearly all introns undergo co-transcriptional splicing, which occurs with higher efficiency for introns in protein-coding genes than for those in noncoding RNAs. Total intron number and intron position are two predominant features that correlate with co-transcriptional splicing efficiency, and introns with alternative 5' or 3' splice sites are less efficiently spliced. Furthermore, we found that mutations in genes encoding trans-acting proteins lead to more introns with increased splicing defects in nascent RNAs than in mature RNAs, and that introns with increased splicing defects in mature RNAs are inefficiently spliced at the co-transcriptional level. Collectively, our results not only uncovered widespread co-transcriptional splicing in Arabidopsis but also identified features that may affect or be affected by co-transcriptional splicing efficiency.
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Project description:The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome-scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they co-regulate a set of cassette exon events, with the majority showing discordant splicing regulation. hnRNPM discordantly regulated splicing events show a positive correlation with splicing during EMT while concordant splicing events do not, highlighting the antagonistic role of hnRNPM and ESRP1 during EMT. Motif enrichment analysis near co-regulated exons identifies guanine-uridine rich motifs downstream of hnRNPM-repressed and ESRP1-enhanced exons, supporting a model of competitive binding to these cis-elements to antagonize alternative splicing. The set of co-regulated exons are enriched in genes associated with cell-migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of co-regulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtypes. In conclusion, hnRNPM and ESRP1 co-regulate antagonistically a set of alternative splicing events that occur during EMT. This regulation is likely mediated through competition for the same intronic binding sites downstream of variable exons. hnRNPM and ESRP1 regulated splicing events are associated with breast cancer survival.
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Project description:Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.
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Project description:To investigate the question that whether Rpb9 affects elongation velocity, thereby indirectly affecting splicing the regulation, we performed time-course GRO-seq after DRB release. In this assay, DRB was first used to block transcription elongation, which caused Pol II to accumulate at TSSs. Upon DRB wash-off, we performed GRO-seq at 0, 10, 20, and 30min to determine the movement of Pol II wave toward gene 3’ end.Based on the hidden Markov model established earlier, we detected a marked reduction of Pol II re-initiation upon DRB removal in response to Rpb9 knockdown (KD) in HeLa cells. This is consistent with the role of Rpb9 in transcription initiation. By determining the mean elongation rate between 10 and 20min and between 20 and 30m, we found accelerated Pol II elongation, as reported earlier, and interestingly, Rpb9 knockdown appeared to have slightly decreased, rather increased, the rate of transcription. We divided all impacted splicing events 4 equal bins and then asked if a higher degree of induced exon skipping tends to occur on genes with faster Pol II movement, finding that this is not the case. Conversely, we divided Rpb9 KD-altered Pol II rate into 4 equal binds and asked whether splicing shows any correlation to Pol II Rpb9 KD-altered Pol II elongation rate, and again, we detected no correlation. We conclude from these data that Rpb9 KD has some minor impact on the Pol II elongation rate in vivo, and importantly, whatever measurable changes in Rpb9 KD-induced Pol II elongation rate are not linked to changes in Rpb9 KD-induced co-transcriptional splicing.
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Project description:In order to measure the impact of U2AF1 on co-transcriptional splicing, we treated HeLa cells with control siRNA or U2AF1 siRNA. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq.
2023-05-27 | GSE175671 | GEO
Project description:In order to measure the impact of Rpb9 on co-transcriptional splicing, we treated HeLa cells with control siRNA or siRpb9, and for comparative analysis, we also depleted Rpb1 and confirmed by Western blotting the knockdown efficiency in all cases. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from each compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility .
2023-11-15 | GSE162235 | GEO
Project description:We examined the role of PTBP1 in regulation of co-transcriptional splicing process by depleting this RNA-binding protein from embryonic stem cells using the auxin-inducible degron technology and analysing the total and chromatin-associated RNA fractions by RNA-seq. We also performed mNET-seq and ChIP-seq analyses using RNA polymerase II- and PTBP1-specific antibodies, respectively. Our data suggest that PTBP1 activates co-transcriptional splicing of hundreds of introns, a surprising effect given that PTBP1 is better known as a splicing repressor. Importantly, some co-transcriptionally activated introns fail to be spliced post-transcriptionally without PTBP1. In a striking example of this regulation, lasting retention of a PTBP1-dependent intron triggers nonsense-mediated decay of mRNAs encoding DNA methyltransferase DNMT3B, explaining their natural expression dynamics in development. Our further analyses suggest that this mechanism may protect differentiation-specific genes from aberrant methylation. We conclude that PTBP1-activated co-transcriptional splicing underlies biologically important decisions.
2023-01-08 | E-MTAB-11544 | biostudies-arrayexpress
Project description:To define the function of Rpb9 on co-transcriptional splicing, we used auxin-inducible Rpb9 degron system in mouse embryonic stem cells (mESCs) with auxin treatment for 3 hours to sufficiently deplete Rpb9. To measure co-transcriptional splicing, we fractionated mESCs cells (spiked in with Drosophila S2 cells for data normalization) into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility.
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