Nuclear Surveillance of long intervening noncoding RNA
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ABSTRACT: Numerous long intervening non-coding RNA (lincRNA) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNA, their synthesis and turnover remain poorly characterised. Here we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal independent Pol II termination on lincRNA as compared to pre-mRNA. In addition, lincRNA are mostly restricted to chromatin where they are co-transcriptionally degraded by the RNA exosome. We also show that a lincRNA specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect functional lincRNA must escape from this targeted nuclear surveillance process.
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.
Project description:RNA polymerase II (Pol II) play an essential role in gene expression. Here, we adapted mammalian Native Elongation Transcript sequencing and Global Run On sequencing to profile nascent RNA genome wide in Arabidopsis. We found Pol II tends to accumulate downstream of transcription start site and pausing at proximal promoter is an important regulatory step for Pol II transcription although loosely controlled. Furthermore, the Pol II with unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream the TSS, while the Ser5P CTD Pol II associates with spliceosome, and the Ser2P CTD Pol II presents a sharp peak 250 base pair downstream of polyadenylation site indicating a stringent control of termination for protein coding genes; whilst the termination of noncoding genes is not. Active expressed genes can be classified into three clusters according to the distribution patterns of different Pol II isoforms. In summary, we demonstrated the modified plant GRO-seq and mNET-seq are suitable to study RNA Pol II dynamics in planta. Although transcription is conserved among high eukaryotes, Pol II has its feature in Arabidopsis.
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.
Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, including SPT5, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. Our phosphoproteomic analyses after CDK9 inhibition, using either DRB or an analog-sensitive CDK9 cell line, confirm the splicing factor SF3B1 as an additional key target of this kinase. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC. As part of this project, we characterized the interactome of SF3B1 in HeLa cells in duplicate.
Project description:RNA polymerase II (Pol II) termination is a crucial step in the transcriptional cycle, ensuring the recycling of Pol II and preventing interference with neighboring gene transcription. Xrn2 is an enzyme with 5'-3'- exonuclease activity, responsible for degrading RNA that is still being transcribed by Pol II after cleavage at the poly(A) site, ultimately leading to Pol II termination. We have confirmed that Xrn2's catalytic activity is essential for timely transcription termination. Furthermore, we have discovered a link between Xrn2 and the conserved, multifunctional transcription factor Spt5. Importantly, our research demonstrates that Xrn2 interacts with Pol II/Spt5 complexes. Additionally, when Spt5 is depleted, it not only impairs global transcription but also results in defective transcription termination. On the other hand, absence of Spt5 leads to the derepression of non-coding transcription and premature termination/attenuation at the 5'-end of coding genes. We propose that Spt5 acts as a "licensing" factor, ensuring that Pol II complexes are properly assembled for efficient transcription and co-transcriptional pre-mRNA processing.
Project description:RNA polymerase II (Pol II) termination is a crucial step in the transcriptional cycle, ensuring the recycling of Pol II and preventing interference with neighbouring gene transcription. Xrn2 is an enzyme with 5'-3'- exonuclease activity, responsible for degrading RNA that is still being transcribed by Pol II after cleavage at the poly(A) site, ultimately leading to Pol II termination. We have confirmed that Xrn2's catalytic activity is essential for timely transcription termination. Furthermore, we have discovered a link between Xrn2 and the conserved, multifunctional transcription factor Spt5. Importantly, our research demonstrates that Xrn2 interacts with Pol II/Spt5 complexes. Additionally, when Spt5 is depleted, it not only impairs global transcription but also results in defective transcription termination. On the other hand, absence of Spt5 leads to the derepression of non-coding transcription and premature termination/attenuation at the 5'-end of coding genes. We propose that Spt5 acts as a "licensing" factor, ensuring that Pol II complexes are properly assembled for efficient transcription and co-transcriptional pre-mRNA processing.
Project description:CDK9 is a critical kinase required for the productive transcription of protein-coding genes by RNA polymerase II (pol II) in higher eukaryotes. Phosphorylation of targets including the elongation factor SPT5 and the carboxyl-terminal domain (CTD) of RNA pol II allow the polymerase to pass an early elongation checkpoint (EEC), which is encountered soon after initiation. In addition to halting RNA polymerase II at the EEC, CDK9 inhibition also causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, suggesting that CDK9 and PP2A, working together, regulate the coupling of elongation and transcription termination to RNA maturation. Our phosphoproteomic analyses, using either DRB or an analog-sensitive CDK9 cell line confirm the splicing factor SF3B1 as an additional key target of this kinase. CDK9 inhibition causes loss of interaction of splicing and export factors with SF3B1, suggesting that CDK9 also helps to co-ordinates coupling of splicing and export to transcription.
Project description:We investigated whether RNA Pol II mediated transcription was altered in Carm1 KO compared to control KO tumor cells. We examined p-Ser2 CTD Pol II relative to total CTD Pol II using mammalian native elongating transcript sequencing (mNET-Seq) (Nojima et al., 2016). Inactivation of the Carm1 gene substantially increased the normalized read density of p-Ser2 CTD Pol II tags relative total Pol II tags. These data provide evidence for altered transcriptional regulation in Carm1 deficient cells.
Project description:The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3’ mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Project description:The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3’ mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.