内含子中polyA位点选择在血管衰老中的作用及调控机制

Vascular aging is the common basis of diverse age-related diseases. Senescence of vascular endothelial cells, smooth muscle cells and fibroblasts is one of the key contributors to vascular aging and functional deterioration. Our previous publications discovered that alternative polyadenylation mediated 3′ UTR lengthening acted as a novel mechanism in regulating senescence of vascular cells (Genome Research 2018; Aging 2019). We recently found mitochondrial located glutaminase coding gene GLS showed reduced usage of intronic polyadenylation during senescence of vascular endothelial cells, smooth muscle cells and fibroblasts. Knocking down GLS transcript generated by intronic polyA site led to senescence-associated phenotypes. We further screened RNA binding protein CPSF6 as one of the upstream regulator for GLS’s intronic polyA site selection. We thus hypothesized that downregulation of GLS’s intronic polyadenylation contributes to senescence of vascular cells. In this proposal, we plan to construct gene-editing mice with specifically knockout of its intronic polyadenylation exon and verify its impact on cellular senescence in diverse tissues especially vessel and heart. The alteration of mitochondrial functions will also be studied. We further investigate the molecular mechanism of CPSF6 in regulating GLS’s intronic polyadenylation and the conservation of such regulation. This study aims to reveal the hidden layer of post-transcriptional regulation for vascular aging, which will enrich the understanding of organ aging.

血管衰老是多种衰老相关疾病的共同基础，而内皮细胞、平滑肌细胞和成纤维细胞的衰老是血管衰老及功能退化的重要原因。我们前期发现选择性多聚腺苷酸化可在血管内皮细胞、平滑肌细胞和成纤维细胞中调控基因3′非翻译区变长并促进细胞衰老（Genome Res.2018;Aging2019）。最新发现线粒体相关谷氨酰胺酶GLS编码基因在血管内皮细胞、平滑肌细胞和成纤维细胞衰老体系中均出现内含子中polyA(pA)位点使用比例下降，特异敲低相应转录本导致细胞衰老相关表型，且筛选出RNA结合蛋白CPSF6可调控该位点选择。据此提出“内含子中pA位点调控血管细胞衰老”假说。后续拟构建GLS内含子pA位点特异敲除鼠，并研究其对小鼠各器官特别是心血管细胞衰老及线粒体功能等影响。同时在人细胞和小鼠中研究CPSF6调控GLS内含子pA位点的机制及保守性。项目从新角度揭示血管衰老中隐藏的遗传调控方式，丰富对器官衰老的理解。

血管衰老是多种衰老相关疾病的共同基础，而内皮细胞、平滑肌细胞和成纤维细胞的衰老是血管衰老及功能退化的重要原因。本培育项目旨在回答申请人提出的“内含子中polyA位点调控血管细胞衰老”的假说。经过三年的研究，本项目圆满完成了研究目标。验证了在细胞衰老中包括GLS基因等内含子中polyA位点的动态变化，及其对细胞衰老的影响，阐明了下调GLS短转录本影响细胞衰老的分子机制，明确了CPSF6调控GLS基因内含子中polyA位点选择的生物学意义，并研发了从全基因组水平发现内含子polyA位点及动态变化的新方法。这一发现从内含子命运决定的新角度提供了细胞衰老调控的新机制，也为后续延缓细胞衰老提供了潜在新靶点。项目发表了6篇标注项目号的相关研究论文（Genome Research 2021; Nucleic Acids Research 2021a,b; Aging Cell 2020; Molecular Oncology 2021; Frontiers in Genetics 2020），均为最后通讯论文和第二标注，其中3篇为内含子polyA位点的方法学及应用的论文，3篇为细胞衰老的内含子及相关基因调控的论文。在本项目的培育下，申请人在2022年12月23日获批了器官衰老与器官退行性变化的机制的集成项目，为成果的后续进一步研发和应用打下了良好基础。