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

项目摘要：尽管骨骼肌中广泛存在选择性剪接，但非常重要的是， 已经研究了通过选择性剪接产生的几种肌肉特异性蛋白质同种型。相反，改变 转录剪接和剪接调节子的表达经常在肌营养不良和衰老相关的 肌肉功能和新陈代谢下降。健康人的骨骼肌占体重的40% 成年人，并在调节全身代谢中起主导作用。然而，替代蛋白质的作用 骨骼肌功能和代谢中的选择性剪接产物在很大程度上是未知的。 我最近的工作表明，Rbfox家族的RNA结合蛋白是至关重要的调节骨骼肌， 成年期的肌肉稳态成年小鼠骨骼肌中的诱导型Rbfox敲除导致约50%的减少 在四周内肌肉质量，改变葡萄糖代谢，剪接>740个基因转录。许多 RBFOX调控的替代外显子在进化上是保守的，提示了替代蛋白的作用 同种型在成人骨骼肌功能中的作用RBFOX蛋白调节细胞膜上相互排斥的E11和E12外显子。 MEF 2D转录因子产生主要的成人骨骼肌特异性同种型MEF 2D β 2。 MEF 2（MEF 2A-D）是高度保守的转录因子家族的四个成员， 胚胎肌肉发育，但其在成人骨骼肌中的作用尚不清楚。E12外显子包含 出生后增加至>75%，以在成人骨骼肌中产生主要的MEF 2D β 2同种型。以确定 为了研究MEF 2D的作用，我使用CRISPR-Cas9删除了Mef 2d的E2外显子，产生了Mef 2d E2 Eko小鼠系。 与野生型小鼠相比，Mef 2d Eko 2小鼠表现出跑步能力和肌肉脂肪酸减少， 氧化 我们的初步数据表明，Mef 2d Eko的肌肉转录组变化极小或无变化。 小鼠我们还发现，大多数MEF 2D存在于骨骼肌的胞质组分中，并与 线粒体和肌肉代谢蛋白。考虑到骨骼肌中肌肉脂肪酸氧化的减少， 在MEF 2D β 2 Eko小鼠中，我们假设MEF 2D β 2蛋白与细胞溶质中的代谢蛋白相互作用， 优化成人骨骼肌中的脂肪氧化。在aim 1中，我们将鉴定和验证与 MEF 2D特异性地或优先地在体内抑制MEF 2D β 2。在aim 2中，我们将确定MEF 2D β 2的损失对 它的相互作用和肌肉脂肪酸氧化，并验证了人类骨骼肌中最重要的MEF 2D β 2-蛋白质相互作用 肌肉组织 骨骼肌葡萄糖和脂肪酸代谢的中断通常在发育前表现出来。 II型糖尿病和肥胖症是现代世界最流行的生活方式疾病之一。因此，我们的工作 将确定MEF 2D β 2在肌肉代谢中的一个新的非经典作用，我们希望这一作用是保守的。 在进化中，MEF 2D的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.