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Transcription elongation rate affects nascent histone pre-mRNA folding and 3’ end processing

ABSTRACT: We report that slow transcription by mutant RNA pol II caused accumulation of polyadenylated histone mRNAs that extend past the stem loop processing site. UV irradiation, which decelerates pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3’ processing by slow pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem loop fails to fold in transcripts made by slow pol II, thereby explaining the absence of SLBP and failure to process 3’ ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure, and suggest a mechanism by which alternative processing could be controlled

ORGANISM(S): Homo sapiens

PROVIDER: GSE109652 | GEO | 2018/02/23

REPOSITORIES: GEO

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Alternative RNA Stuctures Formed During Transcription Depend on Elongation Rate and Modify RNA processing

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.

2021-02-09 | GSE149018 | GEO

A member of transcription/export complex (TREX), THOC5 controls elongation rate by recruiting CDK12.

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.

2022-12-31 | GSE166115 | GEO

Anti-SLBP RIPchip data

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.

2015-12-16 | E-GEOD-59915 | biostudies-arrayexpress

ALYREF is shared by polyadenylated and nonpolyadenylated mRNA processing and export pathways [iCLIP-seq]

Project description:we try to investigate the binding of ALYREF and NXF1 on histone mRNA when the cell treated with indicated siRNAs. ALYREF plays key roles in nuclear export of polyadenylated mRNAs and also modulates their 3' processing, but whether it is involved in regulating RNAs beyond polyadenylated mRNAs is unknown. The replication-dependent (RD) histone mRNAs are not polyadenylated, but end in a stem-loop (SL) structure. Here we demonstrate that ALYREF prevalently binds a region next to the SL on RD histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and ensures this binding. To examine how SLBP KD impact ALYREF distribution on the histone mRNA, we carried out ALYREF iCLIP in control and SLBP KD cells. To investigate the functional consequence for ALYREF binding on histone mRNAs, we isolated polyA+ and polyA- RNAs from control and ALYREF KD cells, and carried out RNA-seq separately.

2019-02-27 | GSE124735 | GEO

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.

2022-08-09 | PXD035866 |

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate

Project description:The Pol II elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, Spt6- and FACT-depleted cells exhibit processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6- depleted strains, but it is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

2024-07-08 | GSE262747 | GEO

mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription

Project description:mRNA cap addition occurs early during RNA pol II transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription, independently of mRNA capping and translation. In cells, sub-lethal suppression of RNMT-RAM reduces RNA pol II occupancy, net mRNA synthesis and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independently of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA-binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription associated factors including RNA pol II subunits, SPT4, SPT6 and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2B K120 ubiquitination, H3 K4 and K36 methylation, RNA pol II S5 and S2 phosphorylation and PAFc recruitment. These findings suggest that multiple interactions between RNMT-RAM, RNA pol II factors and RNA along the transcription unit stimulate transcription.

2018-05-01 | GSE87767 | GEO

THOC5 complexes with other proteins (DDX5, DDX17 and CDK12) essential in primitive cell survival to regulate R loop structures and RNA elongation rate.

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

2022-12-12 | PXD035092 | Pride

Anti-SLBP RIPchip data

2015-12-16 | GSE59915 | GEO

POINT technology illuminates the processing of polymerase-associated intact nascent transcripts

Project description:Mammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular, we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore, POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.

2021-03-19 | GSE159326 | GEO

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