Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilize RNA polymerase I and III complex integrity

Walker-Kopp, Nancy; Jackobel, Ashleigh J.; Pannafino, Gianno; Morocho, Paola A; Xu, Xia; Knutson, Bruce A.

doi:10.1093/hmg/ddx317

Cited by 23 publications

(24 citation statements)

References 47 publications

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“…Overall, these findings are in agreement with previous work using the homologous yeast Pol III, and/or Pol I and Pol II enzymes to map disease-causing mutations 8 – 10 , 15 , 39 . However, the availability of a high-resolution structure of human Pol III enable the comprehensive mapping and rationalization of these allele variants with high confidence.…”

Section: Resultssupporting

confidence: 92%

Structure of human RNA polymerase III

et al. 2020

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In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.

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Section: Resultssupporting

confidence: 92%

Structure of human RNA polymerase III

et al. 2020

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“…Overall, these findings are in agreement with previous work using the homologous yeast Pol III and/or Pol I and Pol II enzymes to map disease-causing mutations [8][9][10]15,37 .…”

Section: Pathologic Genetic Mutations Map To Pol III Subunit Interfacessupporting

confidence: 92%

Structure of human RNA Polymerase III

Ramsay

Abascal-Palacios

Daiß

et al. 2020

Preprint

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ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.

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“…Similarly, a study in X. tropicalis showed that failure to ubiquitylate the ribosome biogenesis factors NOLC1 and TCOF1 destabilizes these proteins, and subsequently disrupts neural crest formation during cell differentiation (Werner et al 2015). Tcof1 is causally linked to the developmental disorder Treacher Collins syndrome (Dixon et al 2006) and involved in rDNA transcription and rRNA modification (Lin and Yeh 2009;Larsen et al 2014;Walker-Kopp et al 2017). While these changes do not appear to affect overall ribosome number or global translation, they moderately affect the translation of a subset of mRNAs encoding proteins involved in neural crest formation (Werner et al 2015).…”

Section: Posttranscriptional Modification Of Rrnamentioning

confidence: 99%

Does functional specialization of ribosomes really exist?

Ferretti

Karbstein

2019

RNA

109

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It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gainof-function assays and the demonstration of inducibility under physiological conditions.

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Treacher Collins syndrome mutations in Saccharomyces cerevisiae destabilize RNA polymerase I and III complex integrity

Cited by 23 publications

References 47 publications

Structure of human RNA polymerase III

Structure of human RNA polymerase III

Structure of human RNA Polymerase III

Does functional specialization of ribosomes really exist?

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