Biology subjects

Ramsay, E. P.

Publications and source records attributed to Ramsay, E. P..

3 recordsLinked to original sources

bioRxiv2024

Structural insights into distinct mechanisms of RNA polymerase II and III recruitment to snRNA promoters

RNA polymerase III (Pol III) is specialized in the transcription of short, essential RNAs, including the U6 small nuclear RNAs (snRNAs). At U6 snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc) forming, together with a Brf2-containing TFIIIB complex, a transcriptionally competent pre-initiation complex (PIC). Additionally, SNAPc is responsible for the recruitment of Pol II at the remaining snRNAs genes (U1, 2, 4 and 5), representing a unique example of a multi subunit transcription factor shared among different RNA Polymerases. The mechanism of SNAPc cross-polymerase engagement and the role of the SNAPC2 and SNAPC5 subunits in transcription remain poorly defined. Here, we present cryo-EM structures of the full-length SNAPc-containing Pol III PIC assembled on the U6 snRNA promoter in the open and melting states at 3.2-4.2[A] resolution. Comparative structural analysis revealed unexpected differences with the yeast PIC and revealed the molecular basis of selective and structurally distinct SNAPc engagement within Pol III and Pol II PICs. Harnessing crosslinking mass spectrometry, we also localize the SNAPC2 and SNAPC5 subunits in proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.

biochemistry↗

bioRxiv2020

Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-[A] resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

biochemistry↗

bioRxiv2020

Structure of human RNA Polymerase III

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.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗