Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures

Proc Natl Acad Sci U S A. 2022 Jan 25;119(4):e2109708119. doi: 10.1073/pnas.2109708119.

Abstract

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

Keywords: Pus7; RNA modification; TruD; pseudouridine; structure.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence*
  • Binding Sites*
  • Catalytic Domain
  • Models, Molecular*
  • Protein Binding
  • Protein Conformation*
  • Protein Interaction Domains and Motifs
  • RNA, Fungal / chemistry
  • RNA, Fungal / genetics
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Stress, Physiological
  • Structure-Activity Relationship
  • Substrate Specificity
  • Temperature
  • Thermodynamics

Substances

  • Pus7 protein, S cerevisiae
  • RNA, Fungal
  • RNA, Messenger
  • Saccharomyces cerevisiae Proteins