MSI1 RNA-binding Modulation Assay

NIH3T3 cells (ATCC CRL-1658)) were co-transfected with the indicated pmiRGLO 3′ UTR reporter plasmid along with either wild-type murine MSI1-eGFP, MSI1-bm-eGFP (which has three phenylalanine to leucine mutations within the first RNA recognition motif (RRM1, F63L/F65L/F68L) that attenuates target RNA association^{37 (link)}), MSI1-AA-eGFP (which is mutated to substitute the two sites of regulatory serine phosphorylation to non-phosphorylatable alanine residues), or eGFP (peGFP N1 empty vector control) plasmids as described previously ^{20 (link),33 (link),34 (link),38 (link),50 (link)}. Expression of the MSI1-eGFP, MSI1-bm-eGFP, and eGFP proteins was confirmed by fluorescence microscopy. Luciferase activity was determined in quadruplicate after 24 h, using the Dual-Luciferase Reporter Assay System (Promega, E2920) and Turner Biosystems luminometer (Promega) according to the supplier's protocol. Data are expressed as relative luciferase activity (FLuc/RLuc) in arbitrary units. All experiments were repeated on at least 3 separate occasions.