Abstract：
The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.
摘要：
编码和非编码因素的复杂相互作用控制着肌肉的生长和发育。这里,我们报道了长链非编码RNA（lncRNA）H19和MyoD（肌源性决定基因1号）在肌肉过程中的相互激活。我们成功克隆了山羊H19的两种同工型，它们在骨骼肌或分化的肌肉卫星细胞（MuSC）中与MyoD转录物显着富集并呈正相关。为了系统地筛选被H19改变的基因，我们使用分化H19缺陷MuSC的cDNA文库进行RNA-seq，并因此锚定MyoD作为介导H19功能的关键基因。有趣的是，MyoD和H19的一些转录物在细胞质中重叠，当核心互补核苷酸突变时，它被严重破坏。同时,MyoDRNA在MS2-RIP实验中成功地拉下H19。此外，HuR可以结合H19和MyoD转录本，而H19或其截短的突变体成功地稳定了MyoDmRNA，有或没有HuR缺乏症。反过来,在H19基因的启动子和外显子中鉴定了新的功能性MyoD蛋白结合位点。我们的结果表明MyoD转录激活H19，RNA-RNA杂交对于H19促进的MyoD表达至关重要，这扩展了我们对肌肉生长中监管网络层次结构的了解。
