The Role Of RBM5 In Alternative Splicing

In metazoan, most transcripts synthesized by RNA polymerase II carry non-coding intronic sequences that must be precisely removed by splicing to generate translatable messenger RNAs (mRNAs). Alternative splicing, a process to remove introns in different combinations, produces diverse mature mRNAs encoding distinct protein isoforms from a single gene. Therefore, accuracy, efficiency and regulation of splicing play critical roles in the modulation of gene expression in higher eukaryotes. RBM5 was originally characterized as candidate tumor suppressor gene in lung cancer. Elevated RBM5 expression inhibits cell proliferation by inducing apoptosis and cell cycle arrest in G1 phase. While RBM5 has the capacity to regulate alternative splicing of apoptosis-related genes, including Fas receptor, c-FLIP and caspase-2, underlying mechanisms remain largely elusive. In order to gain more insights in the biological function of RBM5 in alternative splicing, we performed following studies.1. Regulation of hAID mRNA alternative splicing by RBM5Activation Induced Deaminase (AID) was originally identified as a factor specifically expressed in active B cells. However, abnormal AID expression was also reported in several malignant tumors, such as B cell lymphomas, lung cancers and colorectal cancers. Interestingly, mRNA alternative splicing has recently been suggested to function in the regulation of AID expression. To investigate the mechanism of AID splicing regulation, we constructed a minigene in which a genomic DNA fragment containing exon 3/4/5 was inserted downstream to a CMV promoter. We found that RBM5 overexpression promotes AID exon 4 skipping in the minigene. Furthermore, our data showed that RBM5-mediated exon skipping requires a weak 3'splice site of intron 3. Gel mobility shift assays showed that recombinant RBM5 can bind immediately upstream of the 3'splice site of intron 3 in AID transcripts. The data present here suggest that RBM5 promotes human AID pre-mRNA exon 4 skipping and that this is most likely due to the interference of RBM5 on the recognition of 3'splice site by spliceosome.2. Purification and identification of RBM5-associated protein complexSplicing regulation is generally achieved through RNA-RNA, RNA-protein, or protein-protein interaction. To shed new light on the function of RBM5 in splicing regulation, affinity purification strategy was employed to purify RBM5-containing protein complexes from HeLa nuclear extract. DHX15 and PRP19, two versatile splicing factors, were identified as new RBM5-associated partners. Interaction between RBM5 and PRP19 were further confirmed by co-immunoprecipitation and in vitro GST pull-down assays. While yeast ortholog of DHX15 have been suggested to function in spliceosome disassembly and rRNA biogenesis, the biological function of DHX15 in mammalian cells remains largely unclear. We thus focused on characterization of the interaction between RBM5 and DHX15 in this study.Yeast two-hybrid and co-immunoprecipitation assays showed that the carboxyl-terminal of RBM5 is required for the interaction with DHX15. Point mutation analysis indicated that G patch domain in the carboxyl terminus of RBM5 is indispensable for the interaction between RBM5 and DHX15. Finally, we provide the first evidence that recombinant DHX15 has an ATP-dependent helicase activity. Interestingly, recombinant RBM5 stimulates DHX15 helicase activity in vitro, suggesting of a potentially biological significance of the physical interaction between RBM5 and DHX15. Taken together, these findings provide new insights on the mechanisms by which RBM5 functions as a splicing regulator.