The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression

Duval, Maxime; Yague-Sanz, Carlo; Turowski, Tomasz W.; Petfalski, Elisabeth; Tollervey, David; Bachand, François

doi:10.1038/s41467-023-38826-6

Cited by 6 publications

(7 citation statements)

References 62 publications

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“…Furthermore, we observe a significant increase of reads within RNA Pol I and III transcribed gene loci in samples with sarkosyl indicating a release of paused RNA polymerase I and III complexes (Figure 6 H, I). Pausing has been described to support processing of rRNA co-transcriptionally via the control of RNA polymerase I progression in yeast as this is important for ribosome assembly (60)(61)(62). We conclude that we found further evidence for the existence of RNA polymerase pausing, although the mechanism of RNA polymerase pausing by RNA Pol II and especially for RNA Pol I and III in trypanosomes remains unclear.…”

Section: Rna Polymerase Pausingsupporting

confidence: 64%

Genome-wide Quantification of Polycistronic Transcription inLeishmania major

Grünebast,

Lorenzen,

Clos

2023

Preprint

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Leishmania majoris a human-pathogenic, obligate parasite and the etiological agent of the most prevalent, cutaneous form of leishmaniasis, which is an important neglected, tropical disease with ∼1.2 Mio new infections per year.Leishmania, and the whole order Trypanosomatida, are early eukaryotes with highly diverged gene expression and regulation pathways, setting them apart from their mammalian hosts and from most other eukaryotes. Using precision run-on sequence analysis, we performed a genome-wide mapping and density analysis of RNA polymerases in isolated nuclei of the protozoan parasiteLeishmania major. We map transcription initiation sites within the chromosomes and correlate them with known sites of chromatin modifications. We confirm continuous, polycistronic RNA synthesis in all RNA polymerase II-dependent gene arrays but find varying RNA polymerase activities in polycistronic transcription units (PTUs), excluding gene-specific transcription regulation, but not PTU-specific variations as possible targets of modulatory pathways. Lastly, we find evidence for transcriptional pausing of all three RNA polymerase classes, hinting at a possible mechanism of transcriptional regulation.Significance StatementLeishmaniaspp. are pathogens of humans and animals and cause one of the most important neglected tropical diseases. Regulation of gene expression inLeishmaniabut also in the relatedTrypanosomais radically different from all eukaryotic model organisms, dispensing with regulated, gene-specific transcription, and relying instead on highly regulated translation. Our work sheds light on the initiation, elongation and termination of transcription, maps unidirectional, polycistronic transcription units, provides evidence for transcriptional pausing at or near starting points of RNA synthesis, and quantifies the varying transcription rates of the polycistronic transcription units. Our results will further the understanding of these important pathogens and should provide a valuable ressource for researchers in the field of eukaryotic microbiology.

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Section: Rna Polymerase Pausingsupporting

confidence: 64%

Genome-wide Quantification of Polycistronic Transcription inLeishmania major

Grünebast,

Lorenzen,

Clos

2023

Preprint

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“…Similar to other RNA polymerases, the Pol I elongation process is based on Brownian ratchet in which energydriven mechanism favors forward rather than backward steps. As reported by several studies, Pol I is prone to frequent pausing events and/or regions with slower elongation rates possibly accompanied by backtracking (Clarke et al, 2018;Dangkulwanich et al, 2013;Duval et al, 2023;Guajardo and Sousa, 1997;Turowski et al, 2020a). The dynamic of Pol I elongation is influenced by various factors, such as the folding of the nascent rRNA as well as the supercoiling of the rDNA template, as shown by biophysical modeling recapitulating Pol I density along the gene Turowski et al, 2020a).…”

Section: Introductionmentioning

confidence: 74%

Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription

Azouzi,

Schwank,

Queille

et al. 2023

Preprint

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The RNA polymerase I (Pol I) enzyme that synthesizes large rRNA precursors, exhibits high rate of pauses during elongation, indicative of a discontinuous process. We propose here that Premature Termination of Transcription (PTT) by Pol I is a critical regulatory step limiting rRNA productionin vivo. The Pol I mutant, SuperPol (RPA135-F301S), produces 1.5-fold more rRNA than the wild type (WT). Combined CRAC and rRNA analysis link increased rRNA production in SuperPol to reduced PTT, resulting in shifting polymerase distribution toward the 3’ end of rDNA genes.In vitro, SuperPol shows defective nascent transcript cleavage. Notably, SuperPol is resistant to BMH-21, a drug impairing Pol I elongation and inducing proteasome-mediated degradation of Pol I subunits. Compared to WT, SuperPol maintains subunit stability and sustains high transcription levels upon BMH-21 treatment. These comparative results show that PTT is alleviated in SuperPol while it is stimulated by BMH-21 in WT Pol I.

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“…Peptides were then extracted using acetonitrile, lyophilized, and resuspended in 0.1% trifluoroacetic acid (TFA) to desalt on ZipTips. ZipTips (EMD Millipore) cleanup of peptide samples was performed as described previously ( 56 ).…”

Section: Methodsmentioning

confidence: 99%

Repression of pervasive antisense transcription is the primary role of fission yeast RNA polymerase II CTD serine 2 phosphorylation

Boulanger,

Haidara,

Yague-Sanz

et al. 2024

Nucleic Acids Research

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The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3′ end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.

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The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression

Cited by 6 publications

References 62 publications

Genome-wide Quantification of Polycistronic Transcription inLeishmania major

Genome-wide Quantification of Polycistronic Transcription inLeishmania major

Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription

Repression of pervasive antisense transcription is the primary role of fission yeast RNA polymerase II CTD serine 2 phosphorylation

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