Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. Homo sapiens

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578 leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-mRNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia. Overall design: In order to understand how RBM15 regulates RNA metabolism, we used a leukemia cell line (MEG-01 cell) to establish stable cell lines with RBM15 knockdown. For RNA immunoprecipitation (RIP) assay, normal immunoglobulin was used as controls for sample 1,2; and anti-RBM15 antibody was used for samples 3,4.

RBM15作为一种RNA结合蛋白，可调控包括血液在内的多种组织的细胞命运特化。本研究证实，RBM15的第578位精氨酸残基（R578）可被蛋白质精氨酸甲基转移酶1（protein arginine methyltransferase 1，PRMT1）催化甲基化，随后通过E3泛素连接酶（E3 ligase）CNOT4介导的泛素化途径发生降解。
在急性巨核细胞白血病细胞系中过表达PRMT1，可通过下调RBM15的蛋白水平，阻断巨核细胞的终末分化；而恢复RBM15的蛋白浓度，则可挽救由PRMT1过表达所阻断的巨核细胞终末分化过程。
在分子层面，RBM15可结合巨核细胞生成相关基因的前体mRNA内含子区域，例如GATA1、RUNX1、TAL1及c-MPL。此外，RBM15对特定内含子区域的偏好性结合，可将剪接因子SF3B1招募至相同位点，参与调控可变剪接过程。
因此，PRMT1可通过降低RBM15的蛋白浓度，调控RNA可变剪接；靶向PRMT1或可成为恢复急性巨核细胞白血病患者巨核细胞分化能力的治愈性治疗策略。
实验整体设计：为阐明RBM15调控RNA代谢的具体机制，本研究以白血病细胞系MEG-01为模型，构建了RBM15敲低的稳定细胞系。在RNA免疫沉淀（RNA immunoprecipitation，RIP）实验中，以正常免疫球蛋白（IgG）作为样本1、2的对照；样本3、4则使用抗RBM15抗体进行实验。