Characterization of multimeric complexes formed by the human PTB1 protein on RNA.
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 gamma2 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.
Clerte
Caroline
C
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Hall
Kathleen B
KB
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005091:Exons; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics
D020413:3' Untranslated Regions; D046911:Macromolecular Substances; D020033:Protein Isoforms; D012322:RNA Precursors; D012367:RNA, Viral; D011963:Receptors, GABA-A; D038941:Polypyrimidine Tract-Binding Protein; D012313:RNA