Mechanisms and consequences of 3'UTR isoform diversity in erythropoiesis

项目摘要 成熟信使RNA（mRNA）的3'非翻译区（3' UTR）是终止子与终止子之间的序列。 编码序列的密码子和poly（A）尾。重要的是，3 '端加工机械的位置 将poly（A）尾加到前体mRNA上不是不变的，而是以受控的方式变化以产生3 'UTR 同一基因的mRNA之间的异构体多样性。这个过程被称为选择性多聚腺苷酸化 （阿帕）。虽然通过阿帕产生的3 'UTR同种型不改变蛋白质的氨基酸序列，但它可以使蛋白质的氨基酸序列发生改变。 蛋白质，它们通过添加或去除microRNA和RNA结合位点来影响表达 蛋白质（RBP）影响mRNA的输出，稳定性，定位和翻译效率。靶向3 '端 测序技术表明阿帕在组织之间和特定组织中广泛存在并受到调节， 疾病背景。尽管阿帕流行，但对哪些限制性商业惯例驱动这一过程的监管理解 仍然有限。发现特定造血谱系的细胞显示普遍的阿帕，但没有 存在红系谱系中阿帕的综合图谱。其他RNA加工事件，如替代 已知剪接和翻译控制对于红细胞生成是重要的，并且在某些细胞中失调。 贫血和地中海贫血，这表明阿帕改变3 'UTR的长度/身份也可能影响 健康和疾病中的红细胞生物学。该项目旨在通过全面的方法填补这一知识空白， 在红细胞生成过程中使用靶向3 '末端测序对从以下组织收集的RNA进行鉴定和定量阿帕： 红细胞在整个分化过程中。初步数据表明，有几个基因对红细胞生成至关重要， 如转录因子TAL 1（SCL）和TCF 3（E2 A），在此过程中经历阿帕移位。功能 通过监测对mRNA和蛋白质水平的影响来评估不同3 'UTR同种型选择的影响 （荧光素酶测定）和分化功效（CRISPR缺失以迫使同种型表达）。最后 该项目将通过分析大量的 来自K562红白血病细胞的RBP敲低、突变和敲除实验，随后 实验验证初步数据表明剪接因子在骨髓增生异常中通常发生突变 SRSF 2等综合征（MDS，一种部分以无效红细胞生成为特征的病症）也影响 在红系分化中观察到阿帕变化。总的来说，本提案概述的研究将 提供了对阿帕在红细胞生成过程中使用的新的调节机制的深入了解， 关键基因鉴定这一过程的分子调节剂，其中一些已经涉及到 疾病，可能提出新的治疗途径。

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.