title: Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain.
abstract: Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB21QW, United Kingdom.
authors.forename: Panagiota
authors.initials: P
authors.lastname: Kafasla
authors.forename: Helen
authors.initials: H
authors.lastname: Lin
authors.forename: Stephen
authors.initials: S
authors.lastname: Curry
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Jackson
mesh_terms: D001665:Binding Sites; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010849:Picornaviridae / Q000235:genetics* / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism
chemical_list: D012367:RNA, Viral; D038941:Polypyrimidine Tract-Binding Protein