Identify a new non-canonical role of MEF2D⍺2 protein isoform in skeletal muscle metabolism

项目摘要：尽管选择性剪接在骨骼肌中广泛存在，但非常重要的作用 通过选择性剪接产生的几种肌肉特异性蛋白质亚型已经被研究过。相比之下，改变了 转录剪接和剪接调节因子的表达常见于肌肉营养不良和衰老相关疾病 肌肉功能和新陈代谢下降。骨骼肌占健康人体重的40% 成体，在调节全身新陈代谢方面起着主导作用。然而，交替蛋白质的作用 选择性剪接产物在骨骼肌功能和代谢中的作用在很大程度上是未知的。 我最近的工作表明，RBFox RNA结合蛋白家族对调节骨骼至关重要 成年期的肌肉动态平衡。成年小鼠骨骼肌中可诱导的RBFox基因敲除导致约50%的基因缺失 在四周内的肌肉块中，改变了葡萄糖代谢，并剪接了&gt；740基因转录。许多 RBFox调控的替代外显子在进化上是保守的，暗示了替代蛋白的作用 成人骨骼肌功能中的异构体。Rbfox蛋白调节⍺1和⍺2外显子的相互排斥 MEF2D转录因子产生主要的成人骨骼肌特异性亚型，MEF2D⍺2。 高度保守的转录因子家族中的四个MEF2(MEF2A-D)成员是重要的 对于胚胎肌肉的发育，但它们在成年骨骼肌中的作用尚不清楚。⍺2外显子包涵体 出生后增加到75%，在成人骨骼肌中产生主要的MEF2D⍺2亚型。要确定 利用MEF2D⍺2的作用，我用CRISPR-CAS9缺失了MEF2D的⍺2-外显子，建立了MEF2D⍺2 EKO小鼠系。 与野生型小鼠相比，Mef2d⍺2 EKO小鼠的跑步能力和肌肉脂肪酸水平都有所下降 氧化。 我们的初步数据表明，MEF2D⍺2 EKO肌肉转录组的变化很小，甚至没有变化 老鼠。我们还发现，大多数MEF2D存在于骨骼肌的胞浆部分，并与 线粒体和肌肉代谢蛋白。鉴于骨骼肌中肌肉脂肪酸氧化的减少 MEF2D⍺2 EKO小鼠，我们假设MEF2D⍺2蛋白与胞浆中的代谢蛋白相互作用 优化成人骨骼肌的脂肪氧化。在aim1中，我们将识别和验证与之相互作用的蛋白质 MEF2D-⍺-2可在体内特异性或优先表达。在AIM2中，我们将确定失去MEF2D⍺2对 它的相互作用与肌肉脂肪酸氧化和验证人类骨骼中TOP MEF2D⍺2蛋白的相互作用 肌肉组织。 骨骼肌葡萄糖和脂肪酸代谢的紊乱通常在发育之前就表现出来。 II型糖尿病和肥胖症是现代世界最普遍的生活方式疾病之一。因此，我们的工作 将确定MEF2D⍺2在肌肉代谢中的一个新的非典型作用，我们预计这一作用是保守的 在进化过程中，作为MEF2D，⍺2外显子和剪接位点从鱼类到人类都是保守的。

Project Summary: Despite the widespread occurrence of alternative splicing in skeletal muscle, the role of very few muscle-specific protein isoforms produced by alternative splicing has been studied. In contrast, altered transcript splicing and splicing regulator expression is frequently found in muscle dystrophies and aging-associated decline in muscle function and metabolism. Skeletal muscle makes up to 40% of body weight in healthy human adults and plays a predominant role in regulating whole-body metabolism. Yet, the role of alternate protein products of alternative splicing in skeletal muscle function and metabolism is largely unknown. My recent work demonstrated that the Rbfox family of RNA-binding proteins is vital for regulating skeletal muscle homeostasis in adulthood. Inducible Rbfox knockout in adult mouse skeletal muscle caused ~50% reduction in muscle mass within four weeks, altered glucose metabolism, and splicing of >740 gene transcripts. Many RBFOX-regulated alternative exons are evolutionarily conserved, suggesting roles for the alternate protein isoforms in adult skeletal muscle function. RBFOX proteins regulate mutually exclusive ⍺1 and ⍺2 exons of the MEF2D transcription factor to produce the predominant adult skeletal muscle-specific isoform, MEF2D⍺2. The four MEF2 (MEF2A-D) members of the highly conserved family of transcription factors are important for embryonic muscle development, but their role in the adult skeletal muscle is not known. The ⍺2 exon inclusion increases to >75% after birth to produce the predominant MEF2D⍺2 isoform in adult skeletal muscle. To determine the role of MEF2D⍺2, I deleted the ⍺2-exon of Mef2d using CRISPR-Cas9 to generate, Mef2d⍺2 Eko mouse line. Compared to wild-type mice, Mef2d ⍺2 Eko mice displayed reduced running capacity and muscle fatty acid oxidation. Our preliminary data indicate minimal to no change in muscle transcriptome in muscles of Mef2d⍺2 Eko mice. We also found that most MEF2D is present in the cytosolic fraction of skeletal muscle and interacts with mitochondrial and muscle metabolic proteins. Given the reduced muscle fatty acid oxidation in skeletal muscles of Mef2d⍺2 Eko mice, we hypothesize that MEF2D⍺2 protein interacts with metabolic proteins in the cytosol to optimize fatty oxidation in adult skeletal muscle. In aim1, we will identify and validate proteins interacting with MEF2D⍺2 exclusively or preferentially in vivo. In aim2, we will determine the impact of the loss of MEF2D⍺2 on its interactors and muscle fatty acid oxidation and validate top MEF2D⍺2-protein interactions in human skeletal muscle tissues. A disruption in skeletal muscle glucose and fatty acid metabolism often manifests before the development of type II diabetes and obesity, one of the most prevalent lifestyle diseases of the modern world. Thus, our work will identify a new non-canonical role of MEF2D⍺2 in muscle metabolism, which we expect to be conserved across evolution as MEF2D⍺2 exon and splice sites are conserved from fish to humans.