Efficient RNA polymerase II pause release requires U2 snRNP function

Caizzi, Livia; Monteiro-Martins, Sara; Schwalb, Björn; Lysakovskaia, Kseniia; Schmitzová, Jana; Sawicka, Anna; Chen, Ying; Lidschreiber, Michael; Cramer, Patrick

doi:10.1016/j.molcel.2021.02.016

Cited by 61 publications

(83 citation statements)

References 132 publications

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“…Meanwhile, Pol II ChIP-seq captures the Pol II occupancy on the DNA template in steady state, which depends on the number of polymerases and their elongation velocity (Ehrensberger et al , 2013). Thus, by combining Pol II occupancy measurements with TT-seq derived initiation frequencies, Pol II elongation velocities can be derived (Caizzi et al , 2021). Since Pol II S5p (CTD Serine-5 phosphorylation) appears at TSS after the pre-initiation complex dissociation (Søgaard & Svejstrup, 2007; Glover-Cutter et al , 2009), its signal corresponds most closely to the fraction of productive Pol II amongst the total chromatin-engaged Pol II molecules (Steurer et al , 2018).…”

Section: Resultsmentioning

confidence: 99%

“…The resulting elongation velocities for 3703 genes were used as the “measured elongation velocity” for cross-validation. Elongation velocities v can also be estimated from the ratio of the number of polymerases released into elongation, as measured by TT-seq, over the Pol II occupancy (Gressel et al , 2019; Caizzi et al , 2021). Thus, to derive “estimated elongation velocity” from our multi-omics data we combined TT-seq LRNA coverage with Pol II S5p MINUTE ChIP-seq coverage as follows: This approximation allows relative comparison between different conditions as long as the numerator and denominator terms, TT-Seq and Pol II S5p signals are quantitative.…”

Section: Methodsmentioning

confidence: 99%

“…Such velocity estimations still leave details of global and local elongation dynamics unaddressed (Jonkers & Lis, 2015). Furthermore, it has been shown that with a multi-omics approach, which combines TT-seq with mNET-seq derived Pol II occupancies, transcription elongation velocities can be estimated (Caizzi et al, 2021). Accordingly, we used here the ratio of TT-seq nascent RNA and Pol II S5p coverage as an approximation of elongation velocity and show that it provides a universal and transcriptome-wide measurement of several key parameters: First, we were able to estimate the unperturbed elongation velocity coverage continuously from initiation to termination (Fig EV3C ).…”

Section: Previous Estimates Of Transcription Elongation Kinetics Rely On Chemical Inhibition and Time-seriesmentioning

confidence: 99%

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Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Shao

Kumar

Liedschreiber

et al. 2021

Preprint

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Unique transcriptomes define naïve, primed and paused pluripotent states in mouse embryonic stem cells. Here we perform transient transcriptome sequencing (TT-seq) to de novo define and quantify coding and non-coding transcription units (TUs) in different pluripotent states. We observe a global reduction of RNA synthesis, total RNA amount and turnover rates in ground state naïve cells (2i) and paused pluripotency (mTORi). We demonstrate that elongation velocity can be reliably estimated from TT-seq nascent RNA and RNA polymerase II occupancy and observe a transcriptome-wide attenuation of elongation velocity in the two inhibitor-induced states. We also discover a relationship between elongation velocity and termination read-through distance. Our analysis suggests that steady-state transcriptomes in mouse ES cells are controlled predominantly on the level of RNA synthesis, and that signaling pathways governing different pluripotent states immediately control key parameters of transcription.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Previous Estimates Of Transcription Elongation Kinetics Rely On Chemical Inhibition and Time-seriesmentioning

confidence: 99%

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Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Shao

Kumar

Liedschreiber

et al. 2021

Preprint

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“…On the other hand, the process of splicing has long been suggested to stimulate transcription, although the mechanisms underlying this process remain unclear. Various reports implicate splicing in stimulation of transcription initiation, pause-release, or through the suppression of premature termination [9][10][11][12][13][14] . Notably, a splicing-independent role for the 5' splice site (5'SS) has been suggested 6,8,10,[15][16][17] , and the U1 snRNP can bind to both a 5'SS and RNAPII without the full spliceosome present 18 .…”

Section: Introductionmentioning

confidence: 99%

“…Searches for sequence motifs that could convey these signals using MEME 36 and Homer 37 identified the same top hit enriched among the most abundant TSS-proximal mRNA inserts: a 5' splice site (5'SS) motif. The 5'SS and splicing generally have been suggested to stimulate RNA production [8][9][10][11][12][13][14][15][16][17] , and the mechanisms underlying this activity are currently a subject of great interest 18 . Thus, we used INSERT-seq to systematically assess the effect of intronic sequences on RNA production.…”

Section: Co-transcriptionally Spliced Introns Boost Transcriptionmentioning

confidence: 99%

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential

Vlaming

Martin

et al. 2021

Preprint

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Organismal growth and development rely on RNA Polymerase II (RNAPII) synthesizing the appropriate repertoire of messenger RNAs (mRNAs) from protein-coding genes. Productive elongation of full-length transcripts is essential for mRNA function, however what determines whether an engaged RNAPII molecule will terminate prematurely or transcribe processively remains poorly understood. Notably, despite a common process for transcription initiation across RNAPII-synthesized RNAs, RNAPII is highly susceptible to termination when transcribing non-coding RNAs such as upstream antisense RNAs (uaRNAs) and enhancers RNAs (eRNAs), suggesting that differences arise during RNAPII elongation. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in uaRNAs and eRNAs, rather than specific sequence motifs, underlies the propensity for RNAPII termination on these transcripts. Further, we demonstrate that 5' splice sites exert both splicing-dependent and autonomous, splicing-independent stimulation of transcription, even in the absence of polyadenylation signals. Together, our results reveal a potent role for transcribed sequence in dictating gene output at mRNA and non-coding RNA loci, and demonstrate the power of INSERT-seq towards illuminating these contributions.

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Keeping the balance: The noncoding RNA 7SK as a master regulator for neuron development and function

Briese

Sendtner

2021

BioEssays

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The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis. K E Y W O R D S7SK, heterogeneous nuclear ribonucleoprotein (hnRNP), positive transcription elongation factor b (P-TEFb), survival motor neuron (SMN) This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Efficient RNA polymerase II pause release requires U2 snRNP function

Cited by 61 publications

References 132 publications

Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential

Keeping the balance: The noncoding RNA 7SK as a master regulator for neuron development and function

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