Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

Girbig, Mathias; Misiaszek, Agata D.; Vorländer, Matthias K.; Lafita, Aleix; Grötsch, Helga; Baudin, Florence; Bateman, Alex; Müller, Christoph W.

doi:10.1038/s41594-020-00555-5

Cited by 67 publications

(119 citation statements)

References 76 publications

(114 reference statements)

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“…This is remarkably different in comparison to similar structures of RNA polymerases in elongating states. In the structures of elongating human Pol III, only 5 38 to 6 17 nucleotides were visible, while in yeast Pol I between 7 and 13 nucleotides could be traced 28 . To our surprise, cryo-EM density bearing features of double-stranded RNA could be observed in the exit tunnel (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…In addition, Pol I and Pol III have stably integrated subunits homologous to general transcription factors of Pol II, namely TFIIE and TFIIF 14 . Pol I is the second largest among the eukaryotic RNA polymerases, both in terms of size and the number of subunits, with Pol II consisting of 12 subunits 15,16 and Pol III comprising 17 subunits 17,18 . While yeast Pol I comprises 14 subunits 19,20 , in humans only homologs of 13 subunits have been identified 21 .…”

Section: Introductionmentioning

confidence: 99%

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Cryo-EM structures of human RNA polymerase I

Misiaszek

Girbig

Baudin

et al. 2021

Preprint

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RNA polymerase I (Pol I) specifically synthesizes ribosomal RNA. Pol I upregulation is linked to cancer, while mutations in the Pol I machinery lead to developmental disorders. Here, we report the cryo-EM structure of elongating human Pol I at 2.7 Å resolution. In the exit tunnel, we observe a double-stranded RNA helix that may support Pol I processivity. Our structure confirms that human Pol I consists of 13 subunits with only one subunit forming the Pol I stalk. Additionally, the structure of human Pol I in complex with the initiation factor RRN3 at 3.1 Å resolution reveals stalk flipping upon RRN3 binding. We also observe an inactivated state of human Pol I bound to an open DNA scaffold at 3.3 Å resolution. Lastly, the high-resolution structure of human Pol I allows mapping of disease-related mutations that can aid understanding of disease etiology.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Cryo-EM structures of human RNA polymerase I

Misiaszek

Girbig

Baudin

et al. 2021

Preprint

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“…Recently published structures of human RNAPIII revealed a high level of conservation (Ramsay et al, 2020;Li et al, 2021;Wang et al, 2021). Moreover, mutations of specialized RNAPs lead to genetic disorders, such as Treacher-Collins syndrome and hypomyelinating leukodystrophy (Ramsay et al, 2020;Girbig et al, 2021), demonstrating the requirement for precise coordination among all three RNAPs and their assembly. Research on RNAPIII assembly in yeast focused on rpc128-1007 mutations that disturbed the interface between the Rpc128 and Rpc40 subunits.…”

Section: Discussionmentioning

confidence: 99%

“…Research on RNAPIII assembly in yeast focused on rpc128-1007 mutations that disturbed the interface between the Rpc128 and Rpc40 subunits. Interestingly, multiple disease-associated mutations of human RNAPIII subunits tend to cluster within the region of the RNAPIII assembly platform, suggesting that defects in RNAPIII biogenesis may have severe health consequences (Ramsay et al, 2020;Girbig et al, 2021). Further studies of RNAP assembly should reveal additional factors that are involved in this process and improve our understanding of this vital pathway.…”

Section: Discussionmentioning

confidence: 99%

Specific Features of RNA Polymerases I and III: Structure and Assembly

Turowski

Boguta

2021

Front. Mol. Biosci.

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RNA polymerase I (RNAPI) and RNAPIII are multi-heterogenic protein complexes that specialize in the transcription of highly abundant non-coding RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA). In terms of subunit number and structure, RNAPI and RNAPIII are more complex than RNAPII that synthesizes thousands of different mRNAs. Specific subunits of the yeast RNAPI and RNAPIII form associated subcomplexes that are related to parts of the RNAPII initiation factors. Prior to their delivery to the nucleus where they function, RNAP complexes are assembled at least partially in the cytoplasm. Yeast RNAPI and RNAPIII share heterodimer Rpc40-Rpc19, a functional equivalent to the αα homodimer which initiates assembly of prokaryotic RNAP. In the process of yeast RNAPI and RNAPIII biogenesis, Rpc40 and Rpc19 form the assembly platform together with two small, bona fide eukaryotic subunits, Rpb10 and Rpb12. We propose that this assembly platform is co-translationally seeded while the Rpb10 subunit is synthesized by cytoplasmic ribosome machinery. The translation of Rpb10 is stimulated by Rbs1 protein, which binds to the 3′-untranslated region of RPB10 mRNA and hypothetically brings together Rpc19 and Rpc40 subunits to form the αα-like heterodimer. We suggest that such a co-translational mechanism is involved in the assembly of RNAPI and RNAPIII complexes.

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“…Recent Pol III structural analysis suggests that interactions between the conserved C-terminal tail regions of POLR3G and POLR3GL and the active center of Pol III may function as an autoinhibitory mechanism that precludes transcription in the absence of direct interactions with TFIIIB 52 . While potential differences in POLR3G- and POLR3GL-mediated autoinhibition require further investigation, additional structural insight suggests that subunit POLR3G (RPC7α), but not POLR3GL (RPC7β), may block the site of MAF1 interaction required for repression of Pol III activity 53 . These findings imply that POLR3G and POLR3GL may not be strictly redundant, and that complex identity may indeed exert some level of context-specific control of Pol III transcription potential.…”

Section: Introductionmentioning

confidence: 99%

A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

Bortle

Marciano

et al. 2021

Preprint

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Dysregulation of the RNA polymerase III (Pol III) transcription program, which synthesizes tRNA and other classes of small noncoding RNA critical for cell growth and proliferation, is associated with cancer and human disease. Previous studies have identified two distinct Pol III isoforms defined by the incorporation of either subunit POLR3G (RPC7α) during early development, or POLR3GL (RPC7β) in specialized tissues. Though POLR3G is re-established in cancer and immortalized cell lines, the contributions of these isoforms to transcription potential and transcription dysregulation in cancer remain poorly defined. Using an integrated Pol III genomic profiling approach in combination with in vitro differentiation and subunit disruption experiments, we discover that loss of subunit POLR3G is accompanied by a restricted repertoire of genes transcribed by Pol III. In particular, we observe that a specific class of small noncoding RNA, SNAR-A, is exquisitely sensitive to the availability of subunit POLR3G in proliferating cells. Taken further, large-scale analysis of Pol III subunit expression and downstream chromatin features identifies concomitant loss of POLR3G and SNAR-A activity across a multitude of differentiated primary immune cell lineages, and conversely, coordinate re-establishment of POLR3G expression and SNAR-A features in a variety of human cancers. These results altogether argue against strict redundancy models for subunits POLR3G and POLR3GL, and instead support a model in which Pol III identity itself functions as an important transcriptional regulatory mechanism. Upregulation of POLR3G, which is driven by MYC, identifies a subgroup of patients with unfavorable survival outcomes in specific cancers, further implicating the POLR3G-enhanced transcription repertoire as a potential disease factor.

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Cryo-EM structures of human RNA polymerase III in its unbound and transcribing states

Cited by 67 publications

References 76 publications

Cryo-EM structures of human RNA polymerase I

Cryo-EM structures of human RNA polymerase I

Specific Features of RNA Polymerases I and III: Structure and Assembly

A cancer-associated RNA polymerase III identity drives robust transcription and expression of SNAR-A noncoding RNA

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