RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing

Nojima, Takayuki; Rebelo, Kenny; Gomes, Tomás; Grosso, Ana Rita; Proudfoot, Nicholas J.; Carmo‐Fonseca, Maria

doi:10.1016/j.molcel.2018.09.004

Cited by 134 publications

(171 citation statements)

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“…plaNET‐seq co‐purifies splicing intermediates due to co‐transcriptional spliceosome association with RNAPII (Fig A). The splicing intermediates appear as single‐nucleotide sharp peaks at 5′ splicing site (5′SS) and 3′ splicing site (3′SS) and thus can be distinguished from the nascent reads . We detected an increased fraction of splicing intermediate reads corresponding to 5′SS in plaNET‐seq of NRPB2 Y732F compared to NRPB2 WT , while no obvious difference could be detected for 3′ splicing intermediates (Fig B).…”

Section: Resultsmentioning

confidence: 89%

Organismal benefits of transcription speed control at gene boundaries

et al. 2020

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RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto‐immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read‐through transcription. The accelerated transcription mutant NRPB2‐Y732F exhibits increased association with 5′ splice site (5′SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.

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Section: Resultsmentioning

confidence: 89%

Organismal benefits of transcription speed control at gene boundaries

et al. 2020

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“…Moreover, NET-seq in yeast and mammals allowed estimates of the average length of cryptic read-through transcription that allows quantitative analyses of the transcription termination mechanism(19,20). An additional advantage of NET-seq data are insights into co-transcriptional RNA splicing, since part of the spliceosome is co-purified with transcribing RNAPII complexes(15,21). Nascent RNAPII transcription slows down close to exon-intron boundaries in a splicing-dependent manner and is responsible for intragenic RNAPII stalling(15).…”

Section: Introductionmentioning

confidence: 99%

Native Elongation Transcript sequencing reveals temperature dependent dynamics of nascent RNAPII transcription in Arabidopsis

Kindgren

Ivanov

Marquardt

2019

Preprint

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15Temperature profoundly affects the kinetics of biochemical reactions, yet how large molecular 16 complexes such as the transcription machinery accommodate changing temperatures to maintain 17 cellular function is poorly understood. Here, we developed plant native elongating transcripts 18 sequencing (plaNET-seq) to profile genome-wide nascent RNA polymerase II (RNAPII) transcription 19 during the cold-response of Arabidopsis thaliana with single-nucleotide resolution. Combined with 20 temporal resolution, these data revealed transient genome-wide reprogramming of nascent RNAPII 21 transcription during cold, including characteristics of RNAPII elongation and thousands of non-22 coding transcripts connected to gene expression. Our results suggest a role for promoter-proximal 23 RNAPII stalling in predisposing genes for transcriptional activation during plant-environment 24 interactions. At gene 3'-ends, cold initially facilitated transcriptional termination by limiting the 25 distance of read-through transcription. Within gene bodies, cold reduced the kinetics of co-26 transcriptional splicing leading to increased intragenic stalling. Our data resolved multiple distinct 27 mechanisms by which temperature transiently altered the dynamics of nascent RNAPII transcription 28 and associated RNA processing, illustrating potential biotechnological solutions and future focus 29 areas to promote food security in the context of a changing climate. 30 32Changes to ambient temperatures challenge the development and growth of living organisms. While 33 mammals retain a stable body temperature, sessile organisms such as plants continually sense their 34 environment and rely on molecular mechanisms that compensate for temperature changes(1). 35Alterations to the ambient temperature frequently lead to re-programming of the transcriptional 36 output by RNA polymerase II (RNAPII) that reflects steady-state levels of messenger RNAs and non-37coding RNAs in the cell(2,3). Sequence-specific transcription factors controlling the initiation of 38 transcription often shape these responses. However, the significance of mechanisms regulating 39 eukaryotic gene expression after initiation, for example through control of elongation of the nascent 40 RNA chain is increasingly appreciated(4). Genome-wide profiling of transcriptionally engaged 41 RNAPII complexes has identified low-velocity regions of RNAPII elongation at the beginning (i.e. 42promoter-proximal stalling) and the end (i.e. poly-(A) associated stalling) of genes(4,5). Organisms 43 appear to alter the activity of RNAPII at these regions to re-program their transcriptional output to 44 acclimate to temperature changes. The release from promoter-proximal stalling at heat-shock genes 45 facilitates rapid transcriptional induction in response to heat in Drosophila (6), and promoter-proximal 46 stalling is reduced genome-wide when temperatures increase in mammalian cell cultures (7). RNAPII 47 accumulation at gene ends is associated with the mechanism of transcriptional termination(8)...

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“…Chromatin remodelling proteins play a role in alternative splicing by affecting the chromatin state. The nucleosome density (Tilgner et al, 2009;Luco et al, 2010;Zhou et al, 2014) and the histone modification state at exons (Alló et al, 2009;Tilgner et al, 2009;Luco et al, 2010;Enroth et al, 2012;Curado et al, 2015;Chen et al, 2018) have been proposed to define alternative exons for the splicing machinery and to affect the RNA-polymerase phosphorylation level and the transcription rate (Braunschweig et al, 2013;Fu and Ares, 2014;Zhou et al, 2014;Jonkers et al, 2014;Naftelberg et al, 2015;Fong et al, 2017;Nojima et al, 2018). The BRM affects alternative splicing by increasing the Ser5-P CTD state of RNA-polymerase II at alternative exons in HeLa cells (Batsché et al, 2006;Vorobyeva et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…Only BRM-mut expressing cells displayed a reduced RNA pol II CTD accumulation in exon 5 in MAZ compared to control cells (Supplementary Figure 3I). The phosphorylation of CTD at serine 2 (Ser2-P CTD), and in particular at serine 5 (Ser5-P CTD), plays an important role in the dynamics of RNA pol II and splicing (Batsché et al, 2006;Ito et al, 2008;Ip et al, 2011;Nojima et al, 2018). No differences were found in the levels of Ser2-P CTD or Ser5-P CTD at the exons of MYL6 and GADD45A when expressing BRG1 and BRG1mut compared to control cells ( Figures 3D and 3E, lower panel).…”

Section: Expression Of the Swi/snf Atpases Does Not Change The Rna Pomentioning

confidence: 96%

“…It has been proposed that it depends on the phosphorylation state of the RNA pol II and on the modifications in the chromatin template. A higher Ser5-P CTD slows down or even pauses the RNA pol II, allowing for a time window for the splicing machinery to recognise weak splice sites (Batsché et al, 2006;Hirose et al, 2007;Harlen et al, 2016;Hsin and Manley, 2012;Costódio and Carmo-Fonseca, 2016;Garavis et al, 2017;Nojima et al, 2018). Furthermore, a number of histone modifications have been shown to localise with alternative exons and regulate transcription rate (Gundersen and Johnson, 2009;Luco et al, 2010;Hnilicova et al, 2011;Spain and Govind, 2011;Jonkers et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

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The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

Mackowiak

Guo

et al. 2019

Preprint

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BRG1 and BRM are ATPase core subunits of the human SWI/SNF chromatin remodelling complexes. The function of the SWI/SNF complexes in transcriptional initiation has been well studied, while a function in alternative splicing has only been studied for a few cases for BRM-containing SWI/SNF complexes. Here, we have expressed BRG1 in C33A cells, a BRG1 and BRM-deficient cell line, and we have analysed the effects on the transcriptome by RNA sequencing. We have shown that BRG1 expression affects the splicing of a subset of genes. For some, BRG1 expression favours exon inclusion and for others, exon skipping. Some of the changes in alternative splicing induced by BRG1 expression do not require the ATPase activity of BRG1. Among the exons regulated through an ATPase-independent mechanism, the included exons had signatures of high GC-content and lacked a positioned nucleosome at the exon. By investigating three genes in which the expression of either wild-type BRG1 or a BRG1-ATPase-deficient variant favoured exon inclusion, we showed that expression of the ATPases promotes the local recruitment of RNA binding factors to chromatin and RNA in a differential manner. The hnRNPL, hnRNPU and SAM68 proteins associated to chromatin in C33A cells expressing BRG1 or BRM, but their association with RNA varied. We propose that SWI/SNF can regulate alternative splicing by interacting with splicing-RNA binding factor and altering their binding to the nascent pre-mRNA, which changes RNP structure. Author summarySplicing, in particular alternative splicing, is a combinatorial process which involves splicing factor complexes and many RNA binding splicing regulatory proteins in different constellations. Most splicing events occur during transcription, which also makes the DNA sequence, the chromatin state and the transcription rate at the exons important components that influence the splicing outcome. We show here that the ATP-dependent chromatin remodelling complex SWI/SNF influences the interactions of splicing regulatory factors with RNA during transcription on certain exons that have a high GC-content. The splicing on this type of exon rely on the ATPase BRG1 and favour inclusion of alternative exons in an ATP-independent manner. SWI/SNF complexes are known to alter the chromatin structure at promoters in transcription initiation, and have been previously shown to alter the transcription rate or nucleosome position in splicing. Our results suggests a further mechanism for chromatin remodelling proteins in splicing: to change the interaction patterns of RNA binding splicing regulatory factors at alternative exons to alter the splicing outcome. Biegel JA, Busse TM, Weissman BE. SWI/SNF chromatin remodeling complexes and cancer. Am Lynch KW. Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons. :52. Custódio N, Carmo-Fonseca M. Co-transcriptional splicing and the CTD code. Crit Rev Biochem Mol Biol. 2016 Sep;51(5):395-411. De Conti L, Baralle M...

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RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing

Cited by 134 publications

References 54 publications

Organismal benefits of transcription speed control at gene boundaries

Organismal benefits of transcription speed control at gene boundaries

Native Elongation Transcript sequencing reveals temperature dependent dynamics of nascent RNAPII transcription in Arabidopsis

The SWI/SNF subunits BRG1 affects alternative splicing by changing RNA binding factor interactions with RNA

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