The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

Michael J. Law, Andrew J. Rice, Patti Lin, Ite A. Laird-Offringa

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5′ end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5′ side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.

Original languageEnglish (US)
Pages (from-to)1168-1178
Number of pages11
JournalRNA
Volume12
Issue number7
DOIs
StatePublished - 2006
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Fingerprint

Dive into the research topics of 'The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA'. Together they form a unique fingerprint.

Cite this