Spliceosome specifically associates with CTD phospho-serine 5 isoform of RNA polymerase II to promote co-transcriptional splicing
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ XL
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell Of Cervix, Hela Cell
DISEASE(S): Cervical Cancer
SUBMITTER: 
Kenny Rebelo
LAB HEAD: Maria Carmo Fonseca
PROVIDER: PXD008197 | Pride | 2018-10-05
REPOSITORIES: Pride
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Project description:Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.
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Project description:Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, measuring the kinetics of splicing relative to transcription has been hampered by technical challenges. Here, we took advantage of native elongating transcript sequencing (NET-seq) to identify the position of RNA polymerase II (Pol II) when exons become ligated in the newly synthesized RNA. We analyzed Drosophila melanogaster embryos because the genes transcribed initially during development have few and short introns (like yeast genes), whereas genes transcribed later contain multiple long introns (more similar to human genes). Moreover, compared to human, the Drosophila genome is more compact and thus the coverage of NET-seq reads on intragenic regions is higher. We detected spliced NET-seq reads connected to Pol II molecules that were positioned just a few nucleotides downstream of the 3’ splice site. Although the majority of splice junctions were covered by spliced reads, many introns remained unspliced, resulting in a complex range of heterogeneity in splicing dynamics. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. Both canonical and recursive splicing were associated with a higher Pol II density, suggesting a splicing-coupled mechanism that slows down transcription elongation. We further observed that transcription termination was very efficient for isolated genes but that the presence of an overlapping antisense gene was often associated with transcriptional read-through. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.
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Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
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