PRMT9-catalyzed SF3B2 methylation regulates RNA splicing and synapse development

Human genome encodes nine protein arginine methyltransferases (PRMT1-9), which catalyze three types of arginine methylation: monomethylation (MMA), asymmetric dimethylation (ADMA), and symmetric dimethylation (SDMA). These modifications can alter protein-protein and protein-nucleic acid interactions and play critical roles in transcription regulation and RNA metabolism. A few years ago, we characterized the newest member of the PRMT family--PRMT9 as a SDMA modifying enzyme and identified the splicing factor SF3B2 as its methylation substrate, linking its function to pre-mRNA splicing. However, the biological function of PRMT9 and the molecular mechanism by which PRMT9-catalyzed SF3B2 arginine methylation regulates pre-mRNA splicing remain largely unknown. Here, by charactering an intellectual disability patient-derived PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncovered an important function of PRMT9 in neuronal development. We found that G189R mutation completely abolishes PRMT9 methyltransferase activity and destabilizes the protein by promoting its ubiquitination and proteasome degradation. PRMT9 loss in hippocampal neurons alters RNA splicing of ~1800 transcripts, which likely account for the abnormal synapse development and impaired learning and memory observed in the Prmt9 cKO mouse. Mechanistically, we discovered a critical protein-RNA interaction between the arginine 508 (R508) of SF3B2, the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element located upstream of the branch point sequence (BPS). Additionally, we provide strong evidence that supports SF3B2 being the major and likely only substrate of PRMT9, thus highlighting the conserved function of PRMT9/SF3B2 axis in pre-mRNA splicing regulation. To identify differentially expressed and differentially spliced genes regulated by PRMT9, we performed RNA-seq analysis with polyA+ RNA samples prepared from age and gender matched littermates of wild type control and Prmt9 knockout mouse hippocampus tissues. Each group contains RNA samples from hippocampus of three individual mouse.人类基因组编码9种蛋白质精氨酸甲基转移酶（protein arginine methyltransferase, PRMT）家族成员PRMT1至PRMT9，它们可催化三类精氨酸甲基化修饰：单甲基化（monomethylation, MMA）、不对称二甲基化（asymmetric dimethylation, ADMA）以及对称二甲基化（symmetric dimethylation, SDMA）。此类修饰可改变蛋白质-蛋白质与蛋白质-核酸的相互作用，在转录调控与RNA代谢过程中发挥关键调控作用。数年前，我们团队对PRMT家族的最新成员PRMT9进行了功能表征，确定其为一类SDMA修饰酶，并鉴定剪接因子SF3B2为其甲基化底物，由此将PRMT9的功能与前mRNA剪接过程相联系。然而，PRMT9的生物学功能，以及PRMT9催化的SF3B2精氨酸甲基化调控前mRNA剪接的分子机制，目前仍未完全阐明。本研究通过鉴定一例智力障碍患者来源的PRMT9突变体（G189R），并构建Prmt9条件性敲除（conditional knockout, cKO）小鼠模型，揭示了PRMT9在神经元发育中的重要功能。研究发现，G189R突变可完全丧失PRMT9的甲基转移酶活性，并通过促进其泛素化修饰与蛋白酶体降解，导致该蛋白稳定性下降。海马神经元中PRMT9的缺失会改变约1800种转录本的RNA剪接模式，这或许可以解释Prmt9 cKO小鼠中观察到的突触发育异常与学习记忆受损表型。从分子机制层面，我们发现SF3B2的精氨酸508位点（R508，该位点仅由PRMT9催化甲基化）与前mRNA锚定位点之间存在关键的蛋白质-RNA相互作用；该锚定位点是位于分支点序列（branch point sequence, BPS）上游的顺式调控元件。此外，我们提供了强有力的实验证据，证明SF3B2是PRMT9的主要且很可能是唯一的底物，由此凸显了PRMT9/SF3B2信号轴在前mRNA剪接调控中的保守功能。为了鉴定受PRMT9调控的差异表达基因与差异剪接基因，我们以野生型对照与Prmt9敲除小鼠的海马组织制备的polyA+ RNA样本进行了RNA测序（RNA-seq）分析；实验所用小鼠均为年龄与性别匹配的同窝仔鼠，每组均包含3只独立小鼠的海马组织RNA样本。