Ribotrap Analysis of Proteins Associated with FHL3 3'Untranslated Region in Glioma Cells

ObjectiveTo screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3′ untranslated region (3′UTR) in glioma cells.MethodsWestern blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno- precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS.ResultsPCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein.ConclusionPCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.