RNA结合蛋白在骨骼肌分化的可变剪切调控机制

Embryonic stem cells (ESC) with their ability to differentiate into skeletal muscle in culture hold a great potential for replacement therapies. However, directed differentiation of ESC towards skeletal muscle is still a poorly defined process. We previously found that RNA binding protein Rbm24 was up-regulated during skeletal differentiation. Interestingly we found that Rbm24 regulated some muscle-specific splicing events that are involved in skeletal muscle development. Based on these results, we hypothesize that Rbm24 may play an important role in ESC skeletal differentiation. The current proposal is focused on the role of Rbm24 in ESC skeletal differentiation with the aim to understand cellular and the molecular mechanism by which Rbm24 acts during skeletal specification. Firstly, we will generate a Cre-loxp recombinant ESC line in which the expression of Rbm24 can be temporally and specifically induced upon doxycylin (Dox) addition. We will utilize this inducible system to identify the role and mechanism of Rbm24 in ESC lineage differentiation, Next, we will perform a genome-wide analysis of Rbm24-regulated genes by using high throughput RNA sequencing (RNA-seq) technology, followed by bioinformatics analysis to identify Rbm24-regulated alternative splicing events (AS) which occurred in genes essential for the skeletal differentiation. Finally, Rbm24-expressing GFP tagged cells will be transplanted in Mdx injured mouse model for preliminary evaluation of function improvement of muscle injury. This study will provide significant insight to regulatory network in early development and contribute to a better understanding mechanism of ESC skeletal differentiation.

胚胎干细胞（ESC）因可分化为骨骼肌细胞而可用于再生医学，但其分化调控机制尚不明确。我们研究发现RNA结合蛋白Rbm24在骨骼肌组织中高表达，在ESC骨骼肌分化过程中表达上调。本项目着眼于研究Rbm24在ESC骨骼肌分化中的作用机制。利用强力霉素Dox与Cre/loxP组合的可调控诱导表达系统，在小鼠ESC分化过程中诱导Rbm24过表达，探索Rbm24在分化过程中的作用；高通量转录组测序寻找Rbm24调节的信号通路和可变剪接靶点。进一步在肌营养不良Mdx损伤小鼠模型中对Rbm24阳性骨骼肌细胞移植，评价骨骼肌的功能改善情况。最后利用CRISPR/Cas9系统构建稳定敲除Rbm24人源ESC，探索其在人源ESC骨骼肌分化中的作用机制，验证Rbm24调控的可变剪切靶点。研究结果有望在揭示ESC骨骼肌分化调控机制方面获得新突破，同时将为干细胞来源的骨骼肌在肌营养不良症的临床治疗提供新途径。

骨骼肌具有卓越的再生能力，可以在机械或病理损伤后有效地进行自我修复。然而，肌肉再生的调节机制很复杂，尚未完全阐明。可变剪接(AS)是转录后调控的主要机制，许多异常的AS事件已在伴有异常肌肉再生的肌营养不良症患者中被发现。我们研究发现RNA结合蛋白Rbm24在骨骼肌组织中高表达。然而，关于AS与肌肉再生之间的关系尚不清楚。我们构建了Rbm24系统性条件基因敲除小鼠和卫星细胞特异性Rbm24基因敲除小鼠。并且，我们利用心脏毒素(CTX)诱导的肌肉损伤再生修复模型来评估Rbm24缺失对骨骼肌再生的影响。采用全基因组RNA-Seq的方法，检测Rbm24缺失引起的可变剪接事件的变化。在诱导Rbm24敲除的4个月后，Rbm24敲除小鼠出现了肌肉的异常再生现象。在分子机制方面，通过RNA-Seq我们发现Rbm24在成年小鼠肌肉中调节了一系列可变剪接事件，这些可变剪接事件涉及多个生物过程，包括肌生成、肌肉再生和肌肉肥大。使用CTX诱导的损伤模型，我们发现骨骼肌中Rbm24的缺失导致肌源性融合和分化缺陷，并显著延迟肌肉再生。此外，卫星细胞特异性Rbm24基因敲除小鼠再现了在Rbm24系统性基因敲除小鼠中再生缺陷表型。重要的是，我们证明了Rbm24直接调节Mef2d、Naca、Rock2和Lrrfip1的可变剪接，这些基因的可变剪接对成肌分化和肌肉再生至关重要。纠正肌肉营养不良患者中发现的参与肌肉再生的异常剪接事件可能是治疗肌肉营养不良患者的一种新疗法。

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