Mechanisms and consequences of 3'UTR isoform diversity in erythropoiesis

项目摘要 成熟信使RNA(MRNAs)的3‘非翻译区(3’非翻译区)是位于 编码序列的密码子和Poly(A)尾巴。重要的是，3‘端加工机械的位置 将Poly(A)尾巴添加到前mRNA并不是一成不变的，而是以受控的方式发生变化，从而产生3‘UTR 同一基因的mRNAs之间的异构体多样性。这一过程被称为交替多聚腺苷。 (APA)。虽然通过APA产生的3‘UTR亚型不会改变该蛋白的氨基酸序列 蛋白质，它们通过增加或移除microRNAs和RNA结合的结合位点来影响表达 影响信使核糖核酸输出、稳定性、定位和翻译效率的蛋白质(RBPs)。目标3‘端 测序技术表明，APA在组织间和特定组织中广泛存在并受到调节 疾病背景。尽管APA盛行，但对哪些限制性商业惯例推动这一过程的监管理解 仍然是有限的。发现特定造血系的细胞表现为弥漫性APA，但没有 在红系谱系中存在APA的全面图谱。其他RNA处理事件，如Alternative 剪接和翻译控制，已知对红细胞生成很重要，在某些情况下调节失调 贫血和地中海贫血，这表明APA改变3‘UTRs的长度/同源性也可能影响 健康与疾病中的红系生物学。该项目寻求通过全面的 用RNA3‘端测序鉴定和定量红细胞生成过程中的APA 红系细胞在整个分化过程中。初步数据显示，有几个基因对红血球的生成至关重要， 与转录因子TAL1(SCL)和TCF3(E2a)一样，在这一过程中也经历了APA的变化。功能界别 不同的3‘非编码区亚型选择的影响将通过监测对mRNA和蛋白质水平的影响来评估 (荧光素酶分析)和分化效率(CRISPR缺失以强制异构体表达)。最后，这一点 该项目将通过分析一个大型的概要来确定红细胞生成过程中APA变化的关键调节因素 K562红白血病细胞的RBP基因敲除、突变和敲除实验 实验验证。初步数据显示剪接因子在骨髓异常增生症中常见突变 像SRSF2这样的综合征(MDS，一种部分以无效的红细胞生成为特征的疾病)也会影响 在红系分化过程中观察到APA的移位。综上所述，这项建议概述的研究将 洞察APA在红细胞生成过程中的新调节机制，这些机制在功能上发生了变化 关键基因。识别这一过程的分子调节因子，其中一些已经牵涉到 疾病，可能会提出新的治疗途径。

Project Summary The 3’ untranslated region (3’UTR) of mature messenger RNAs (mRNAs) is the sequence between the stop codon of the coding sequence and poly(A) tail. Importantly, the location where the 3’end processing machinery adds the poly(A) tail to the pre-mRNA is not invariant, but changes in a controlled manner to generate 3’UTR isoform diversity between mRNAs of the same gene. This process is known as alternative polyadenylation (APA). Although the 3’UTR isoforms generated through APA do not alter the amino acid sequence of the protein, they influence expression by adding or removing binding sites for microRNAs and RNA binding proteins (RBPs) that influence mRNA export, stability, localization, and translation efficiency. Targeted 3’end sequencing techniques have shown APA to be widespread and regulated between tissues and in specific disease contexts. Despite the prevalence of APA , a regulatory understanding of which RBPs drive this process remains limited. Cells of specific hematopoietic lineages were found to display pervasive APA, but no comprehensive map of APA in the erythroid lineage exists. Other RNA processing events, like alternative splicing and translational control, are known to be important for erythropoiesis and dysregulated in certain anemias and thalassemias, suggesting that APA altering the length/identity of 3’UTRs may also influence erythroid biology in health and disease. This project seeks to fill this knowledge gap by comprehensively identifying and quantifying APA during erythropoiesis using targeted 3’end sequencing on RNA collected from erythroid cells throughout differentiation. Preliminary data suggests several genes essential for erythropoiesis, like transcription factors TAL1 (SCL) and TCF3 (E2A), undergo APA shifts during this process. The functional impact of different 3’UTR isoform choices will be assessed by monitoring impact on mRNA and protein levels (luciferase assays) and differentiation efficacy (CRISPR deletions to force isoform expression). Finally, this project will identify key regulators of the APA shifts across erythropoiesis by analyzing a large compendium of RBP knockdown, mutation, and knockout experiments from K562 erythroleukemia cells followed by experimental validation. Preliminary data suggests splicing factors commonly mutated in myelodysplastic syndromes (MDS, a condition characterized, in part by ineffective erythropoiesis) like SRSF2, also influence APA shifts observed in erythroid differentiation. Taken together, the studies outlined by this proposal will provide insight into novel regulatory mechanisms of APA utilized during erythropoiesis that functionally alters key genes. Identification of the molecular regulators of this process, some of which are already implicated in disease, may suggest novel therapeutic avenues.