Transcription elongation rate affects nascent histone pre-mRNA folding and 3’ end processing
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE109652 | GEO | 2018/02/23
REPOSITORIES: GEO
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Project description:Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing, and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3’ splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary the plasticity of nascent transcripts has widespread effects on RNA processing.
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Project description:THOC5, a member of the THO complex that is a subcomplex of Transcription/export complex (TREX) 1, is essential for 3´processing of slow kinetics IEGs, and for export of only a subset of genes. Applying nanopore mRNA-sequence technology, we show here that upon depletion of THOC5, 50% of mRNAs showed alteration of 3´end cleavage sites. Moreover, polymerase-II (Pol II)-CHIP-sequencing data reveals that upon depletion of THOC5 Pol II density was increased at gene body and 3´UTR. THOC5 is recruited near to Pol II high density sites except promotor regions, suggesting that THOC5 is involved in transcription elongation. Indeed, 385 THOC5 independent genes in fast Pol II cells became THOC5 dependent in slow Pol II cells. Chromatin associated THOC5 in slow Pol II cells interacts with CDK12, RNA helicases DDX5 and DDX, and THOC6, but not with other members of THO complex. Notably, THOC5 did not form this complex in fast poly II cells. CDK12 was recruited to R-loop (DNA:RNA hybrid) in THOC5/THOC6 dependent manner. Here, THOC6, but not THOC5 directly interacts with R-loop then, recruited DDX5 resolves R-loop and then CDK12 enhances further transcription elongation. These data show for the first time the novel function of THOC5/THOC6 complex during transcription elongation.
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Project description:The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. They are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in at a conserved stem-loop (SL) structure. This SL structure interacts stably with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including RNA Binding Protein IP followed by microarray analysis (RIP-ChIP) to analyze the RNA binding landscape of SLBP. Reference based design on two color Agilent DNA microarrays. Experimental sample is labeled in Ch1 (Cy3), which are mRNA bound by the SLBP RNA-binding protein. The reference sample is labeled in Ch2 (Cy5) which are the polyribosomal mRNAs from the supernatant that were not bound by anti-SLBP. Controls included no antibody in the IP (which tends to have non-specific background binding to the beads) and pre-incubation of the SLBP with the antibodies antigenic peptide, which blocks antibody binding to the SLBP.
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Project description:THOC5, a member of the THO complex, is essential for the 3´processing of some inducible genes, the export of a subset of genes and stem cell self-renewal. Utilising nanopore mRNA-sequencing we show that when THOC5 is depleted 50% of mRNAs undergo altered 3´end cleavage. Further, THOC5 depletion leads to increased RNA Polymerase II (Pol II) presence on the gene body and close to the 3’end. Moreover, THOC5 is recruited close to high density Pol II sites except those at the promotor regions suggesting that THOC5 is involved in transcriptional elongation. Indeed, THOC5 independent genes in cells expressing fast Pol II became THOC5 dependent in cells expressing slow Pol II. In cells expressing slow Pol II chromatin associated THOC5 interacts with CDK12 (a protein that modulates mRNA elongation rates), RNA helicases DDX5, DDX17, and THOC6. Importantly these interactions were not observed in cells expressing fast Pol II. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate that THOC5/THOC6 play a part in transcription elongation. Given the role of THOC5 in primitive cell survival its phosphorylation by agonists, oxidative stress and oncogenes the pathway we identified has relevance in the physiology and pathology of stem cells
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