Evidence for two protein coding transcripts at the Igf2as locus

The insulin-like growth factor 2 antisense (Igf2as) gene is part of the Ins-Igf2-H19 imprinted gene cluster. The function of the paternally expressed Igf2as is still elusive.In our previous work, we showed that Igf2as transcripts were located in the cytoplasm of C2C12 mouse myoblast cells, associated with polysomes and polyadenylated suggesting that Igf2as is protein coding. In the present work, the protein coding capacity of Igf2as was investigated. We demonstrate for the first time the existence of a polypeptide translated from an Igf2as construct. Furthermore, an RNA-Seq analysis was performed using RNA prepared from skeletal muscles of newborn wild-type and deltaDMR1-U2 mice to further elucidate the function of Igf2as transcripts. We found no evidence for a regulatory role of Igf2as in the imprinted gene cluster. Interestingly, the RNA-Seq analysis indicated that Igf2as plays a role in the energy metabolism, the cell cycle, histone acetylation and muscle contraction pathways. Our Igf2as investigations further elucidated that there are two distinct Igf2as transcripts corresponding to two putative ORFs. Skeletal muscle tissue from four wild-type and four deltaDMR1-U2 female newborn mice were selected and mRNA expression compared in a RNA-Seq experiment.胰岛素样生长因子2反义（insulin-like growth factor 2 antisense, Igf2as）基因隶属于Ins-Igf2-H19印记基因簇。目前，父本表达的Igf2as的功能仍有待阐明。在前期研究中，我们证实Igf2as转录本定位于C2C12小鼠成肌细胞的细胞质中，可与多聚核糖体结合且存在多腺苷酸化修饰，提示该基因具备蛋白编码潜能。本研究针对Igf2as的蛋白编码能力展开探究，首次验证了由Igf2as表达载体翻译得到的多肽的存在。此外，我们利用新生野生型及deltaDMR1-U2小鼠的骨骼肌总RNA开展RNA测序（RNA-Seq）分析，以进一步解析Igf2as转录本的功能。结果显示，未发现Igf2as在该印记基因簇中发挥调控作用的证据。值得注意的是，RNA测序分析表明，Igf2as参与能量代谢、细胞周期、组蛋白乙酰化及肌肉收缩通路的调控过程。本研究还发现，存在两种截然不同的Igf2as转录本，分别对应两个推定开放阅读框（ORFs）。本次RNA测序实验选取了4只野生型雌性新生小鼠与4只deltaDMR1-U2雌性新生小鼠的骨骼肌组织，对二者的mRNA表达水平进行了对比分析。