LncRNA-Six1编码短肽与MyoD互作调控家鸡肌肉生长的作用机制研究

It's well known that lncRNA could play a regulatory role by acting as a non-coding RNA molecule. In this study, we propose a new model that lncRNA regulates muscle growth, which is that lncRNA encodes a kind of short peptides that interact with transcription factors. In our previous study, we identified a 7.2KD short peptide (Small Six1) encoded by lncRNA-Six1, and found that this peptide is related to myoblast proliferation and differentiation. However, how the peptide Small Six1 regulates muscle growth remains unclear. In this study, we propose that the short peptide Small Six1 could interacts with MyoD. First, by using Co-IP technology, we will validate the interaction between Small Six1 encode by lncRNA-SIX1 and MyoD, and further to investigate the molecular mechanism of interaction. Then, we suppose to discover the crystal structure of Small Six1-MyoD protein complex using protein crystallography method. In this study, we aim to reveal the functional pathway and molecular regulatory networks of MyoD interacting by Small Six1, and further to reveal the new functional model that lncRNA-Six1 regulates muscle growth in chicken. The completion of this study is helpful with scientific significance for identifying of the epigenetic mechanism for muscle growth in animals, and is potentially valuable for the genetic improvement of growth traits for yellow chicken industry.

长链非编码RNA(LncRNA)作为一种非编码的调控性RNA分子来发挥作用已经被人熟知，本项目我们提出LncRNA通过编码短肽与转录因子互作从而调控肌肉生长的新作用模式。我们前期鉴定出由lncRNA-Six1编码的，一个7.2KD的短肽Small Six1，其与成肌细胞增殖分化有关，但是该短肽发挥作用的具体机制仍不清楚。本项目拟以lncRNA-Six1编码的短肽Small Six1结合MyoD为切入点，通过Co-IP等技术，对Small Six1结合MyoD的分子机理进行分析；结合蛋白质晶体学技术，解析Small Six1与MyoD蛋白复合物的晶体结构。本项目旨在明确短肽Small Six1结合MyoD的作用通路和分子调控网络，揭示lncRNA-Six1调控家鸡肌肉生长的新作用模式。本项目的完成对揭示动物肌肉生长的表观遗传机制有重要科学意义，同时对黄羽肉鸡生长性状的遗传改良有潜在应用价值。

项目背景：.我国肉鸡品种主要分为“白羽肉鸡”和“黄羽肉鸡”两大类型。黄羽肉鸡在产肉效率远远低于白羽肉鸡。长链非编码RNA是一类重要的调控性RNA分子，但其在肉鸡产肉性状方面的调控作用，尚不十分清楚。..主要研究内容：.（1）增殖期（GM）和分化期(DM)家鸡原代成肌细胞的lncRNA测序和蛋白质组测序。（2）LncRNA翻译出蛋白质的遗传机理。（3）LncRNA翻译出蛋白质调控成肌细胞增殖和分化的机制。（4）LncRNA在肌肉萎缩中的作用。（5）LncRNA的小ORF瓦解靶基因SMURF2 mRNA的稳定性。（6）LncRNA通过调控TGF-β/SMAD通路，参与肌肉发育。..重要结果：.（1）GM与DM相比较，一共发现了3106个差异表达基因和72个差异表达lncRNA ；（2）GM与DM相比较，蛋白组测序发现了104个能够编码短肽的lncRNAs；（3）Western blot检测发现lncRNA SMUL能够翻译出蛋白质；（4）LncRNA SMUL促进了成肌细胞增殖，抑制了成肌细胞的分化和减少肌管的面积；（5）在活体水平上，利用慢病毒lncRNA SMUL过表载体注射1日龄小鸡的肌肉后，肌纤维的横截面积减少，同时增加了肌肉萎缩标志基因（ATROGIN1, MURF1）的表达量；（6）通过RNA半衰期实验证明，lncRNA SMUL的小ORF通过无义降解途径，抑制了靶基因SMURF2的表达；（7）LncRNA SMUL通过抑制靶基因SMURF2，激活TGF-β/SMAD通路，调控肌肉的生长发育。..关键数据及其科学意义：.关键数据：蛋白组测序发现了104个能够编码短肽的lncRNAs；大部分短肽长度分布在21-40个氨基酸的长度范围；58.04%和27.27%编码短肽的lncRNAs分别来源于外显子lncRNA和基因间lncRNA。科学意义：以往的认识中，lncRNA是不能编码蛋白质的，通过本项目，我们发现一部分lncRNA也能够编码出蛋白质。因此，我们证明了lncRNA的ORF是家鸡蛋白质库的重要组成部分。