Guide RNA acrobatics: positioning consecutive uridines for pseudouridylation by H/ACA pseudouridylation loops with dual guide capacity

Genes Dev. 2022 Jan 1;36(1-2):70-83. doi: 10.1101/gad.349072.121. Epub 2021 Dec 16.

Abstract

Site-specific pseudouridylation of human ribosomal and spliceosomal RNAs is directed by H/ACA guide RNAs composed of two hairpins carrying internal pseudouridylation guide loops. The distal "antisense" sequences of the pseudouridylation loop base-pair with the target RNA to position two unpaired target nucleotides 5'-UN-3', including the 5' substrate U, under the base of the distal stem topping the guide loop. Therefore, each pseudouridylation loop is expected to direct synthesis of a single pseudouridine (Ψ) in the target sequence. However, in this study, genetic depletion and restoration and RNA mutational analyses demonstrate that at least four human H/ACA RNAs (SNORA53, SNORA57, SCARNA8, and SCARNA1) carry pseudouridylation loops supporting efficient and specific synthesis of two consecutive pseudouridines (ΨΨ or ΨNΨ) in the 28S (Ψ3747/Ψ3749), 18S (Ψ1045/Ψ1046), and U2 (Ψ43/Ψ44 and Ψ89/Ψ91) RNAs, respectively. In order to position two substrate Us for pseudouridylation, the dual guide loops form alternative base-pairing interactions with their target RNAs. This remarkable structural flexibility of dual pseudouridylation loops provides an unexpected versatility for RNA-directed pseudouridylation without compromising its efficiency and accuracy. Besides supporting synthesis of at least 6% of human ribosomal and spliceosomal Ψs, evidence indicates that dual pseudouridylation loops also participate in pseudouridylation of yeast and archaeal rRNAs.

Keywords: RNA-guided RNA modification; RNA–RNA interaction; box H/ACA RNAs; guide RNA acrobatics; pseudouridine; pseudouridylation.

MeSH terms

  • Humans
  • Nucleic Acid Conformation
  • Pseudouridine* / chemistry
  • RNA / chemistry
  • RNA, Guide* / chemistry
  • RNA, Guide* / genetics
  • RNA, Ribosomal
  • Uridine

Substances

  • RNA, Guide
  • RNA, Ribosomal
  • Pseudouridine
  • RNA
  • Uridine