Structural dynamics govern substrate recruitment and catalytic turnover in H/ACA RNP pseudouridylation

RNA Biol. 2021 Sep;18(9):1300-1309. doi: 10.1080/15476286.2020.1842984. Epub 2020 Nov 10.

Abstract

H/ACA ribonucleoproteins catalyse the sequence-dependent pseudouridylation of ribosomal and spliceosomal RNAs. Here, we reconstitute site-specifically fluorophore labelled H/ACA complexes and analyse their structural dynamics using single-molecule FRET spectroscopy. Our results show that the guide RNA is distorted into a substrate-binding competent conformation by specific protein interactions. Analysis of the reaction pathway using atomic mutagenesis establishes a new model how individual protein domains contribute to catalysis. Taken together, these results identify and characterize individual roles for all accessory proteins on the assembly and function of H/ACA RNPs.

Keywords: H/ACA complexes; RNP assembly; pseudouridylation; smFRET spectroscopy; structural dynamics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Base Pairing
  • Catalysis
  • Pseudouridine / genetics
  • Pseudouridine / metabolism*
  • Pyrococcus furiosus / genetics
  • Pyrococcus furiosus / metabolism*
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Small Nucleolar / genetics
  • RNA, Small Nucleolar / metabolism*
  • Ribonucleoproteins, Small Nucleolar / genetics
  • Ribonucleoproteins, Small Nucleolar / metabolism*
  • Spliceosomes

Substances

  • Archaeal Proteins
  • RNA, Small Nucleolar
  • Ribonucleoproteins, Small Nucleolar
  • Pseudouridine

Grants and funding

This work was supported by the Deutsche Forschungsgemeinschaft (DE) [SFB902].