Cryo-EM structures of human RNA polymerase I

Misiaszek, Agata D.; Girbig, Mathias; Baudin, Florence; Murciano, Brice; Lafita, Aleix; Müller, Christoph W.

doi:10.1038/s41594-021-00693-4

Cited by 32 publications

(42 citation statements)

References 106 publications

(204 reference statements)

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“…Structural comparisons indicate that eukaryotic polymerases can also accommodate these RNA secondary structures within their RNA exit channels ( 51 ). A very recent structural study on human RNAPI has provided evidence for the presence of double-stranded RNA in the RNA exit channel ( 52 ). Therefore, we propose that, in the case of RNAPIII, the formation of an RNA hairpin can induce destabilizing conformational changes in RNAPIII that would contribute to the dissociation of the EC but only when a T-tract resides in the polymerase main channel (Fig.…”

Section: Discussionmentioning

confidence: 99%

An integrated model for termination of RNA polymerase III transcription

et al. 2022

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RNA polymerase III (RNAPIII) synthesizes essential and abundant noncoding RNAs such as transfer RNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the nontemplate strand. Here, we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

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Section: Discussionmentioning

confidence: 99%

An integrated model for termination of RNA polymerase III transcription

et al. 2022

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“…The K408 and K410 residues are located on top of the clamp module in the RNAP I complex (Figure 3A), a region that participates in the transition between the open and closed conformation of the polymerase 31, 32 . Of note, sequence and structure alignment of yeast Rpa190 and human Rpa194 indicate that K408 is conserved in evolution and is occupied by K350 in the human RNAP I complex (Supplementary Figure 3A and 3B) 33 . Rpa190 has a prominent ubiquitination band, with an electrophoretic mobility equivalent to a mass increase of around 29 KDa, which might reflect the addition of 3 ubiquitin molecules (Figure 3A); besides, there are two extra fainter bands with a mass increase comparable to the addition of 4 and 6 ubiquitin molecules, respectively.…”

Section: Resultsmentioning

confidence: 99%

Ubiquitin proteomics uncovers RNA polymerase I as a critical target of the Nse1 RING domain

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et al. 2021

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Ubiquitination controls numerous cellular processes, and its deregulation is associated to many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and important functions in genome integrity. However, Nse1-dependent ubiquitin targets remain largely unknown. Here, we use label-free quantitative proteomics to analyse the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts on the ubiquitination of several proteins involved in DNA damage tolerance, ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of the Smc5/6 complex in chromosome segregation. In addition, our analysis uncovers an unexpected connection between Nse1 and RNA polymerase I (RNAP I) ubiquitination. Specifically, Nse1 promotes the ubiquitination of K408 and K410 in A190, the largest subunit of RNAP I, to induce its degradation. We propose that this mechanism contributes to Smc5/6-dependent rDNA disjunction in response to transcriptional elongation defects.

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“…The latter is related to Pol II initiation factors TFIIF, and TFIIE ( 15 ) and has homologues in Pol III ( 16 ). The stalk was proposed to be divergent between yeast and human, as DNA- and protein sequence–based searches have not identified a homologue of subunit A14 in human cells and the lack of which was recently confirmed by structural investigations of the human enzyme ( 17 , 18 ). Table 1 summarizes the subunit terminologies for yeast and mammalian Pol I in comparison with human Pol II and Pol III subunits and correlates nomenclature.…”

Section: Introductionmentioning

confidence: 99%

“…Recently published Pol I elongation complex structures showed an increased flexibility of the clamp domain within the human enzyme with additional clamp–DNA contacts present in elongation complex structures, whereas the clamp domain was open and showed increased flexibility in an inactive state ( 17 , 18 ). Furthermore, the stalk subcomplex of human Pol I also shows increased flexibility because of a reduced number of contacts with the Pol I core but shows significant movement upon interaction with initiation factor Rrn3 ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans

et al. 2022

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Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This “dock II” domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor–binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain–containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

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Cryo-EM structures of human RNA polymerase I

Cited by 32 publications

References 106 publications

An integrated model for termination of RNA polymerase III transcription

An integrated model for termination of RNA polymerase III transcription

Ubiquitin proteomics uncovers RNA polymerase I as a critical target of the Nse1 RING domain

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans

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