RBM15-Mediated RNA Splicing Fine-Tunes Epigenetic Program through Interaction with SF3B1

RBM15, an RNA binding protein belongs to the SPEN family, which is evolutionally conserved from plant to mammals. The relevance of RBM15 to blood diseases came to spot light when RBM15 was discovered to be involved in chromosome translocation t(1;22) in acute megakaryoblastic leukemia. RBM15 is indispensible for the self-renewal of hematopoietic stem cells in response to stress and for megakaryocyte terminal differentiation. RBM15 knockdown results in the unchecked generation of megakaryocyte progenitors in mouse models. At the molecular level, RBM15 have been shown to be a transcriptional repressor in NOCTH signaling pathway by recruiting HDAC complexes like another family member SHARP. RBM15 interacts with transcriptional coactivator SET1/ASH2. RBM15 controls the alternative splicing of thrombopoietin receptor (MPL). However, the molecular mechanisms on how RBM15 regulates alternative RNA splicing is less known.

We identified the RBM15-associated proteins with mass spectrometry analysis. Pathway analysis found that RBM15 is mainly associated with proteins involved in RNA splicing, which is also validated by immune-fluorescent microscopy with anti-RBM15 antibody. Among the RBM15-associated proteins are SF3B1 and U2AF, which are heavily mutated in myeloid dysplasia syndrome and chronic lymphocytic leukemia. The interaction between RBM15 and SF3B1 is further validated by co-immunoprecipitation assays. Furthermore, we found that RBM15-SF3B1 interaction is regulated by protein arginine methylation of RBM15. RBM15 is responsible for recruiting SF3B1 complex to the pre-mRNA intronic regions with weak U2snRNP binding sites. We did pair-end, 100bp read RNA-seq of RNA isolated from RBM15 knockdown leukemia cells, and used RBM15 antibody to immunoprecipiate sonicated RNAs to identify intronic regions bound by RBM15. Combining these next generation sequencing data, we found about 50% of the RBM15 binding sites are in the intronic regions and 45% of the RBM15 binding sites are in the 3'UTR regions. RBM15 regulates alternative splicing of a few known transcription factors involved in hematopoiesis such as GATA1, Fli1, GFIb, and RUNX1 as well as chromatin binding proteins such as BRD4, HDAC7 etc.. Therefore, RBM15-SF3B1 interaction fine-tunes the optimal dose of transcription factors needed for the generation of blood lineages in response to environmental stress. How the mutant SF3B1 proteins disrupt its interaction with RBM15 will be discussed.

In addition to RBM15-mediated RNA splicing, we also found that RBM15 per se is regulated by its antisense counterpart (AS-RBM15). AS-RBM15 is a long non-coding RNA whose transcription is activated by RUNX1. AS-RBM15 promotes megakaryocyte differentiation by activating RBM15 protein translation. Therefore, RBM15 and AS-RBM15 are at the center of a RNA regulatory network, which is linked to epigenetic programs regulated by RUNX1 and GATA1. Given that both RUNX1 and SF3B1 are often mutated in myeloid dysplasia syndrome, understanding RBM15-mediated gene regulation offers much needed knowledge for understanding MDS.