title: Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1.
abstract: T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors.forename: William J
authors.initials: WJ
authors.lastname: Bauer
authors.forename: Jason
authors.initials: J
authors.lastname: Heath
authors.forename: Jermaine L
authors.initials: JL
authors.lastname: Jenkins
authors.forename: Clara L
authors.initials: CL
authors.lastname: Kielkopf
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence / Q000502:physiology; D001665:Binding Sites; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D039102:Poly(A)-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D020836:Protein Structure, Quaternary; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D000074019:T-Cell Intracellular Antigen-1
chemical_list: D051017:Apoptosis Regulatory Proteins; D039102:Poly(A)-Binding Proteins; D016601:RNA-Binding Proteins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human; D012313:RNA