Characterization of multimeric complexes formed by the human PTB1 protein on RNA

Caroline Clerte, Kathleen B. Hall

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

Polypyrimidine tract binding protein (PTB or hnRNP I) has several known functions in eukaryotic cells, including exon exclusion during alternative splicing events, mRNA stabilization, and regulation of viral translation and replication. PTB contains four RNA Binding Domains (RBDs, or RRMs), all of which can potentially bind RNA, but their roles in the various biological functions of PTB are not clear. We investigate the properties of the complexes formed by human PTB1 on two target RNAs: the rat GABAA receptor γ2 subunit pre-mRNA and the Hepatitis C Virus 3′ NonTranslated RNA. The GABA RNA contains four polypyrimidine tracts in the intron and exon, while the HCV NTR contains a 75-nt U-rich tract and a highly structured 3′-terminus. Electrophoretic mobility shift assays show that PTB1 protein first binds to both RNAs with nanomolar affinities, but subsequent protein addition leads to formation of higher-order complexes. Stoichiometry experiments show that the ultimate complexes contain up to eight PTB1 proteins per RNA strand. Protein constructs containing two tandem RBDs also bind the two RNAs, but with different affinities and stoichiometries. Nuclease protection assays show that PTB1 protects the polypyrimidine tracts in the GABA RNA, as does a construct consisting of RBD3 and RBD4; however, a construct containing RBD1 and RBD2 enhances cleavage of bound RNA. The binding mechanisms of PTB1 are unique to the full-length protein; these modes appear to include direct association with the RNA as well as weaker intermolecular protein associations. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish
Pages (from-to)457-475
Number of pages19
JournalRNA
Volume12
Issue number3
DOIs
StatePublished - Mar 2006

Keywords

  • GABA pre-mRNA
  • HCV RNA
  • PTB1
  • Polypyrimidine tract binding protein

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