SF3B4 ‘reads’ RNA that is methylated by METTL16

Abstract N 6-Methyladenosine (m6A) is a prevalent RNA modification that regulates multiple aspects of RNA metabolism, including RNA localization, stability, decay, and translation. m6A deposition is catalyzed by distinct methyltransferase complexes, including the METTL3/14 complex and METTL16, which recognize different RNA sequence motifs. The biological effects of m6A are mediated by effector proteins that selectively recognize m6A-modified RNA. To identify previously uncharacterized m6A-binding proteins, we developed an RNA-binding protein domain array to systematically screen for candidate m6A effectors. Using this approach, we identified the spliceosomal protein SF3B4 as a potential m6A reader. RNA pulldown assays using m6A-modified RNA probes demonstrated selective enrichment of endogenous SF3B4, supporting its ability to recognize m6A-modified RNA. To define the RNA targets of SF3B4, we performed SF3B4 RIP-seq alongside m6A RIP-seq, followed by RIP-qPCR validation of overlapping targets. Motif analysis revealed that SF3B4 preferentially associates with the conserved GRAGRA (R = A/G) motif, consistent with the RNA sequence recognized by the METTL16 methyltransferase. Notably, transcripts of the BCR and MET oncogenes were identified as shared targets of SF3B4 and METTL16. Together, these findings identify SF3B4 as a previously unrecognized m6A effector and suggest that it participates in RNA metabolic processes downstream of METTL16-mediated m6A modification.