细胞骨架调控基因Rras2的选择性多聚腺苷酸化影响细胞衰老的作用和机制

Transcription is the first key step for genetics information delivery in central dogma. Transcription initiation, elongation and termination are three major steps to generate mature messenger RNA (mRNA) as template for translation. RNA processing machinery has significant functional changes in senescent cells compared to younger ones. However, the consequence of such changes remain elusive, especially regarding polyadenylation of mRNA. Alternative polyadenylation (APA) can lead to generation of distinct 3′ untranslated regions (3′ UTR) of mRNA, which affects RNA stability and localization, translation efficiency, thus plays important roles in many biological processes such as development and cancer. Our previous work showed that replicative senescent mouse cells underwent global 3′ UTR lengthening. Those genes have longer 3′ UTRs displayed decreased gene expression level and enriched in cell senescence associated pathways such as cell cytoskeleton regulation. Among them, Rras2 gene is an example. Longer 3′ UTR of Rras2 generates less protein than shorter one. Knockdown of Rras2 leads to senescence related phenotypes. We thus hypothesize that APA leads to cellular senescence via lengthening of 3′ UTR of senescence associated genes. In this proposal, we will further validate the function of APA in human cells and other types of cell senescence models. In addition, whether overexpression of Rras2 gene can reverse senescence related phenotypes will be confirmed. The mechanism under which longer 3′ UTR generates less protein will be explored. We will identify the key cis elements and trans-acting factors contributing to the protein production. Finally, which upstream factors (e.g., RNA binding proteins) control the global lengthening of 3′ UTR in senescent cells will be screened and validated. This study will deepen our understanding of cellular senescence from APA as a new angle.

细胞衰老是组织及个体衰老的原因之一。选择性多聚腺苷酸化（APA）可决定信使RNA使用不同长度的3′非翻译区（3′UTR），并参与众多生理病理过程，但其是否在细胞衰老中起作用目前尚无报导。我们前期研究发现，小鼠和大鼠细胞衰老中APA可使3′UTR总体变长，相关基因表达量下降，并富集在细胞骨架调控等细胞衰老相关通路。其中Rras2基因长的3′UTR相比短的翻译出更少蛋白质，且敲低该基因引起了显著的细胞衰老表型。据此提出“APA可通过延长基因3′UTR调控细胞衰老”的假说。后续拟以Rras2基因为切入点，在多个细胞衰老体系中验证APA的作用，阐明APA影响细胞衰老的分子机制，确定控制长的3′UTR翻译蛋白变少的关键顺式作用元件、反式作用因子，以及衰老细胞中调控APA的上游因子，为理解细胞衰老乃至个体衰老提供新的研究角度，并为延缓衰老提供新的突破点。

细胞衰老是一种重要的抑癌机制，并且也是个体衰老的原因之一。之前的研究发现可变多聚腺苷酸化(APA)介导的mRNA 3ʹ UTR总体变短在癌细胞中可上调基因表达并和表型相关，但APA在细胞衰老中的变化规律及其功能仍不清楚。我们利用PA-seq方法发现几百个基因在衰老细胞中倾向于使用远端pA位点，从而使得3ʹ UTR总体变长，并下调基因的稳态表达量。有意思的是，这些3ʹ UTR变长的基因富集在多个细胞衰老相关的通路中。我们随后对候选基因Rras2进行了深入的功能和机制研究。Rras2基因属于Ras超家族成员并在多个信号转导通路中起重要作用。Rras2基因在小鼠、大鼠和人的多个细胞衰老体系中均呈现了3ʹ UTR变长的现象，并且长的3ʹ UTR可引起蛋白质产量的下降。而敲低Rras2基因可引起众多细胞衰老相关表型。过表达Rras2可部分回复相应细胞表型。深入的机制研究发现剪接因子TRA2B结合在可变3ʹ UTR区域（即长的3ʹ UTR相比短的3ʹ UTR所多出的部分）的AGAA基序上对蛋白质的产量下降起到了重要作用。敲低TRA2B或者突变该基序均可部分回复对蛋白质表达量的影响。过表达TRA2B也可下调RRAS2蛋白质水平并促进细胞产生衰老相关表型。值得注意的是，Rras2基因近端和远端pA位点的polyA信号及AGAA基序在多个物种中保守，提示APA介导的基因转录后调控在进化上可能具有重要作用。此外，我们还发现RNA结合蛋白HuR可在上游调控人RRAS2基因的3ʹ UTR长度，并且Rras2表达下调后可引起基因编辑小鼠心脏舒张和收缩末期直径变小，心脏舒张和收缩末期容积下降。同时，秀丽线虫衰老中更多基因3ʹ UTR变长，在秀丽线虫中敲除Rras2同源基因可影响线虫寿命。这些结果提示该基因3ʹ UTR长度调控不仅可以在细胞水平影响衰老，也可以在器官乃至个体水平影响衰老。在Rras2基因的启发下，我们还发现人CDK16基因的3ʹ UTR变长可调控癌细胞衰老，而PTEN及HN1基因的3ʹ UTR长度改变也可调控细胞衰老。