Identification of a noncanonical RNA binding domain in the U2 snRNP protein SF3A1

William Martelly, Bernice Fellows, Kristen Senior, Tim Marlowe, Shalini Sharma

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

During splicing of pre-mRNA, 5' and 3' splice sites are brought within proximity by interactions between the pre-mRNA bound U1 and U2 snRNPs, followed by recruitment of the tri-snRNP for assembly of the mature spliceosome. Previously, we identified an interaction between the U2 snRNP-specific protein SF3A1 and the stem-loop 4 (SL4) of the U1 snRNA that occurs during the early steps of spliceosome assembly. Although harboring many annotated domains, SF3A1 lacks a canonical RNA binding domain. To identify the U1-SL4 binding region in SF3A1, we expressed amino- and carboxy-terminal deletion constructs using a HeLa cell-based cell free expression system. UV-crosslinking of the truncated proteins with 32P-U1-SL4 RNA identified the carboxy-terminal ubiquitin-like (UBL) domain of SF3A1 as the RNA binding region. Characterization of the interaction between SF3A1-UBL and U1-SL4 by electrophoretic mobility shift assay and surface plasmon resonance determined high binding affinity (KD = ∼97 nM), and revealed the double-stranded G-C rich stem of U1-SL4 as an important feature for binding to the UBL domain. Further, mutations of two conserved tyrosine residues, Y772 and Y773, were found to cause a two- and fivefold decrease in the binding affinity for U1-SL4, respectively. Finally, we found that SF3A1-UBL can specifically pull down the U1 snRNP from HeLa nuclear extract, demonstrating its capacity to bind U1-SL4 in the context of the intact snRNP. Thus, the data show that the UBL domain of SF3A1 can function as an RNA binding domain and that mutations in this region may interfere with U1-SL4 binding.

Original languageEnglish (US)
Pages (from-to)1509-1521
Number of pages13
JournalRNA (New York, N.Y.)
Volume25
Issue number11
DOIs
StatePublished - Nov 1 2019

Keywords

  • pre-mRNA splicing
  • SF3A1
  • stem–loop 4
  • U1 snRNA
  • ubiquitin-like domain

ASJC Scopus subject areas

  • Molecular Biology

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