Novel RNA-binding protein P311 binds eukaryotic translation initiation factor 3 subunit b (eIF3b) to promote translation of transforming growth factor ?1-3 (TGF-?1-3).
P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-?1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD ... confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a Kd of 1.26 ?m. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-?1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-? 5'UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-?s 5'UTRs and eIF3b, stimulates the translation of TGF-?1, 2, and 3.
Mesh Terms:
5' Untranslated Regions, Amino Acid Sequence, Animals, Binding Sites, Escherichia coli, Eukaryotic Initiation Factor-3, Gene Expression Regulation, Genes, Reporter, Humans, Luciferases, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, NIH 3T3 Cells, Nerve Tissue Proteins, Oncogene Proteins, Protein Binding, Protein Biosynthesis, Rats, Recombinant Proteins, Sequence Alignment, Signal Transduction, Transforming Growth Factor beta1, Transforming Growth Factor beta2, Transforming Growth Factor beta3
5' Untranslated Regions, Amino Acid Sequence, Animals, Binding Sites, Escherichia coli, Eukaryotic Initiation Factor-3, Gene Expression Regulation, Genes, Reporter, Humans, Luciferases, Male, Mice, Mice, Inbred C57BL, Molecular Sequence Data, NIH 3T3 Cells, Nerve Tissue Proteins, Oncogene Proteins, Protein Binding, Protein Biosynthesis, Rats, Recombinant Proteins, Sequence Alignment, Signal Transduction, Transforming Growth Factor beta1, Transforming Growth Factor beta2, Transforming Growth Factor beta3
J. Biol. Chem.
Date: Dec. 05, 2014
PubMed ID: 25336651
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