An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation.
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ABSTRACT:
SUBMITTER: Hieb AR
PROVIDER: S-EPMC1500975 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transcription of protein-coding genes is regulated by dynamic association of co-factors with RNA polymerase II (RNAPII). The function of these factors and their relationship with RNAPII is often poorly understood. Here, we present an approach for elongation-factor-specific mNET capture (ELCAP) of RNAPII complexes for sequencing and mass spectrometry analysis aimed at investigating the function of such RNAPII regulatory proteins. As proof of principle, we apply ELCAP to the RNAPII-associated proteins SCAF4 and SCAF8, which share an essential role as mRNA anti-terminators but have individual roles at the 3' end of genes. Mass spectrometry analysis shows that both SCAF4 and SCAF8 are part of RNAPII elongation complexes containing 3' end processing factors but depleted of splicing components. Importantly, the ELCAP sequencing (ELCAP-seq) profiles of SCAF4- and SCAF8-RNAPII complexes nicely reflect their function as mRNA-anti-terminators and their competing functions at the end of genes, where they prevent or promote transcriptional readthrough.
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Project description:Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.
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Project description:The elongation phase of transcription by RNA polymerase II (RNAP II) is tightly controlled by a large number of transcription elongation factors. Here we describe experimental approaches for the isolation of RNAPII elongation complexes in vitro and the use of these complexes in the examination of the function of a variety of factors. The methods start with formation of elongation complexes on DNA templates immobilized to paramagnetic beads. Elongation is halted by removing the nucleotides and the ternary elongation complexes are then stripped of factors by a high salt wash. The effect of any factor or mixture of factors on elongation is determined by adding the factor(s) along with nucleotides and observing the change in the pattern of RNAs generated. Association of a factor with elongation complexes can be examined using an elongation complex-electrophoretic mobility shift assay (EC-EMSA) in which elongation complexes that have been liberated from the beads are analyzed on a native gel. Besides being used to dissect the mechanisms of elongation control, these experimental systems are useful for analyzing the function of termination factors and mRNA processing factors. Together these experimental systems permit detailed characterization of the molecular mechanisms of elongation, termination, and mRNA processing factors by providing information concerning both physical interactions with and functional consequences of the factors on RNAPII elongation complexes.
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Project description:Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.
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Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
| S-EPMC6200757 | biostudies-literature
Project description:Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.
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Project description:Multiple RNA polymerase II (RNAPII) molecules can transcribe a gene simultaneously, but what happens when such polymerases collide--for example due to polymerase pausing or DNA damage? Here, RNAPII collision was characterized using a reconstituted system for simultaneous transcription by two polymerases. When progression of leading polymerase is obstructed, rear-end collision entails a transient state in which the elongation complexes interact, followed by substantial backtracking of trailing polymerase. Elongation complexes remain stable on DNA, with their activity and the integrity of transcription bubbles remaining intact. Subsequent TFIIS-stimulated transcript cleavage allows resumed forward translocation, resulting in trailing polymerase oscillating at the obstruction. Conversely, if leading polymerase is merely stalled at a pause site, collision and TFIIS cooperate to drive it through. We propose that dynamic interactions between RNAPII elongation complexes help regulate polymerase traffic and that their conformational flexibility buffers the effect of collisions with objects on DNA, thereby maintaining stability in the face of obstacles to transcription.
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Project description:The transcriptional coactivator Sub1 has been implicated in several steps of mRNA metabolism in yeast, such as the activation of transcription, termination, and 3'-end formation. In addition, Sub1 globally regulates RNA polymerase II phosphorylation, and most recently it has been shown that it is a functional component of the preinitiation complex. Here we present evidence that Sub1 plays a significant role in transcription elongation by RNA polymerase II (RNAPII). We show that SUB1 genetically interacts with the gene encoding the elongation factor Spt5, that Sub1 influences Spt5 phosphorylation of the carboxy-terminal domain of RNAPII largest subunit by the kinase Bur1, and that both Sub1 and Spt5 copurify in the same complex, likely during early transcription elongation. Indeed, our data indicate that Sub1 influences Spt5-Rpb1 interaction. In addition, biochemical and molecular data show that Sub1 influences transcription elongation of constitutive and inducible genes and associates with coding regions in a transcription-dependent manner. Taken together, our results indicate that Sub1 associates with Spt5 and influences Spt5-Rpb1 complex levels and consequently transcription elongation rate.
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Project description:Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.
| S-EPMC10598743 | biostudies-literature
Project description:MotivationSequencing total RNA without poly-A selection enables us to obtain a transcriptomic profile of nascent RNAs undergoing transcription with co-transcriptional splicing. In general, the RNA-seq reads exhibit a sawtooth pattern in a gene, which is characterized by a monotonically decreasing gradient across introns in the 5'-3' direction, and by substantially higher levels of RNA-seq reads present in exonic regions. Such patterns result from the process of underlying transcription elongation by RNA polymerase II, which traverses the DNA strand in a 5'-3' direction as it performs a complex series of mRNA synthesis and processing. Therefore, data of sequenced total RNAs could be utilized to infer the rate of transcription elongation by solving the inverse problem.ResultsThough solving the inverse problem in total RNA-seq has the great potential, statistical methods have not yet been fully developed. We demonstrate what extent the newly developed method can be useful. The objective is to reconstruct the spatial distribution of transcription elongation rates in a gene from a given noisy, sawtooth-like profile. It is necessary to recover the signal source of the elongation rates separately from several types of nuisance factors, such as unobserved modes of co-transcriptionally occurring mRNA splicing, which exert significant influences on the sawtooth shape. The present method was tested using published total RNA-seq data derived from mouse embryonic stem cells. We investigated the spatial characteristics of the estimated elongation rates, focusing especially on the relation to promoter-proximal pausing of RNA polymerase II, nucleosome occupancy and histone modification patterns.Availability and implementationA C implementation of PolSter and sample data are available at https://github.com/yoshida-lab/PolSter.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC6546130 | biostudies-literature