Cellular stress alters 3’UTR landscape through alternative polyadenylation and isoform-specific degradation

Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different lengths of 3’ untranslated region (3’UTR). Here we show arsenic stress elicits global shortening of 3’UTRs through two mechanisms. First, stress leads to immediate shortening of 3’UTR due to preferential usage of proximal cleavage and polyadenylation sites (PASs), as revealed by 3’ end sequencing of newly made RNAs that are metabolically labeled with 4-thiouridine. Second, long 3’UTR isoforms are more rapidly degraded during recovery from stress as compared to short 3’UTR isoforms, further shortening 3’UTR lengths in the cell. Using ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing (3’READS+RIP), we show that the RNA-binding protein T cell-restricted intracellular antigen-1 (Tia1) preferentially interacts with long 3’UTR isoforms via U-rich elements in alternative 3’UTR sequences, and the interaction correlates with stress granule (SG) association during stress and with mRNA decay during recovery from stress, indicating SG-mediated RNA clearance mechanism post stress. Importantly, genes whose 3’UTRs are shortened by APA during stress can evade stress-induced 3’UTR size-based mRNA degradation, leading to higher transcript abundance post stress. Moreover, proliferating and differentiated cells display different extents of 3’UTR shortening after stress, indicating cell type-specific of impact of stress on the 3’UTR landscape. Together, our data indicate that 3’UTR length plays important roles in gene expression in stressed cells, and APA functions as an adaptive stress response mechanism to preserve mRNAs. 4 RNA-seq libraries from NIH3T3 cells for gene expression analysis; 4 3'READS libraries for analysis of APA using total RNA from NIH3T3 cells treated with sodium arsenite for 1h; 6 3'READS libraries for analysis of APA time course using total RNA from NIH3T3 cells treated with sodium arsenite for 1h and recovered for different amount of time; 8 3'READS libraries for analysis of APA isoform stability using 4sU metabolic labeling in NIH3T3 cells; 8 3'READS libraries for analysis of stress response of proliferating and differented C2C12 cells using cytoplasmic RNA; 4 3'READS libraries for the analysis of APA during C2C12 cell differentiation.

绝大多数真核基因均可表达携带不同长度3’非翻译区（3’ untranslated region, 3’UTR）的可变多聚腺苷酸化（alternative polyadenylation, APA）异构体。本研究揭示，砷胁迫可通过两种机制引发细胞内3’UTR的全局性缩短。其一，胁迫会通过优先使用近端切割多聚腺苷酸化位点（proximal cleavage and polyadenylation sites, PASs）快速诱导3’UTR缩短，该结论通过对经4-硫尿苷（4-thiouridine，简称4sU）代谢标记的新生RNA开展3’末端测序得以验证。其二，相较于短3’UTR异构体，长3’UTR异构体在胁迫恢复阶段的降解速率更快，进一步缩短了细胞内3’UTR的整体长度。本研究借助核糖核蛋白免疫沉淀偶联3’末端测序（ribonucleoprotein immunoprecipitation coupled with 3’ end sequencing, 3’READS+RIP）技术发现，RNA结合蛋白（RNA-binding protein）T细胞限制性胞内抗原-1（T cell-restricted intracellular antigen-1, Tia1）可通过可变3’UTR序列中的富含U元件（U-rich elements）优先结合长3’UTR异构体；该结合过程与胁迫阶段的应激颗粒（stress granule, SG）招募以及恢复阶段的mRNA降解均呈显著相关性，提示胁迫后存在应激颗粒介导的RNA清除机制。尤为关键的是，在胁迫期间经APA缩短3’UTR的基因，可规避胁迫诱导的、基于3’UTR长度的mRNA降解途径，最终在胁迫后维持更高的转录本丰度。此外，增殖细胞与分化细胞在胁迫后的3’UTR缩短程度存在显著差异，表明胁迫对3’UTR调控全景的影响具有细胞类型特异性。综上，本研究数据证实，3’UTR长度在胁迫状态下的基因表达过程中发挥核心调控作用，而APA作为一种适应性胁迫应答机制，可实现mRNA的留存与稳态维持。本研究配套的测序文库信息如下：用于基因表达分析的NIH3T3细胞RNA-seq（RNA测序）文库共4套；采用经1h亚砷酸钠（sodium arsenite）处理的NIH3T3细胞总RNA进行APA分析的3’READS文库共4套；采用经1h亚砷酸钠处理并经不同时长恢复培养的NIH3T3细胞总RNA进行APA时间进程分析的3’READS文库共6套；采用经4sU代谢标记的NIH3T3细胞进行APA异构体稳定性分析的3’READS文库共8套；采用胞质RNA对增殖及分化C2C12细胞的胁迫应答进行分析的3’READS文库共8套；用于分析C2C12细胞分化过程中APA变化的3’READS文库共4套。