Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

Background: P311 is a stimulator of TGF-1-3 translation, but its mechanism of action is unknown. Results: P311 binds eIF3b and the 5UTRs of TGF-1-3 mRNAs. Thereby, P311 recruits TGF-1-3 mRNAs to the translation machinery. Conclusion: P311 is an RNA-binding protein that binds to eIF3b to stimulate TGF-1-3 translation. Significance: Our studies add a new level of complexity to TGF- signaling regulation. 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 (R) (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 K-d 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- 5UTRs 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 5UTRs and eIF3b, stimulates the translation of TGF-1, 2, and 3.