Mechanisms and consequences of 3'UTR isoform diversity in erythropoiesis

红细胞生成中 3UTR 亚型多样性的机制和后果

• 批准号: 10705017
• 负责人: Matthew Robert Gazzara
• 金额: $ 5.02万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705017
• 关键词: 3' Untranslated Regions; Acute Erythroblastic Leukemia; Alternative Splicing; Anemia; Automobile Driving; Binding Proteins; Binding Sites; Biological Assay; Biology; Blood; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Data; Development; Differentiation Antigens; Disease; Distal; Dysmyelopoietic Syndromes; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Event; GATA1 gene; Gene Expression; Genes; Genetic Transcription; Goals; Health; Hematopoietic; Human; Immune; Iron; K-562; K562 Cells; Knock-out; Knowledge; Length; Location; Luciferases; Maps; Messenger RNA; MicroRNAs; Mitochondria; Molecular; Monitor; Mutate; Mutation; Northern Blotting; Output; Pattern; Play; Poly A; Poly(A) Tail; Polyadenylation; Prevalence; Process; Protein Isoforms; Proteins; Proteomics; RNA; RNA Processing; RNA Splicing; RNA Stability; RNA-Binding Proteins; Race; Regulation; Role; SRSF2 gene; Site; Specific qualifier value; TAL1 gene; TCF3 gene; Techniques; Terminator Codon; Thalassemia; Time; Tissues; Trans-Activators; Transcript; Translations; Validation; Work; candidate validation; cell type; erythroid differentiation; erythroleukemic cell; experimental study; gene function; insight; knock-down; mutant; novel; novel therapeutics; protein aminoacid sequence; protein expression; stem cells; transcription factor; transcriptome; transcriptome sequencing

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.

项目概要 成熟信使 RNA (mRNA) 的 3' 非翻译区 (3'UTR) 是终止子之间的序列 编码序列的密码子和 Poly(A) 尾部，重要的是 3' 端加工机器的位置。 将 Poly(A) 尾添加到前 mRNA 中并不是不变的，而是以受控方式发生变化以生成 3'UTR 同一基因的 mRNA 之间的异构体多样性被称为选择性多聚腺苷酸化。 (APA) 虽然通过 APA 生成的 3’UTR 亚型不会改变氨基酸序列。 蛋白质，它们通过添加或删除 microRNA 和 RNA 结合的结合位点来影响表达 影响 mRNA 输出、稳定性、定位和翻译效率的蛋白质 (RBP)。 测序技术表明 APA 在组织和特定组织之间广泛存在并受到调节 尽管 APA 很流行，但监管机构对哪些 RBP 驱动了这一过程的了解。 发现特定造血谱系的细胞表现出普遍的 APA，但没有发现。 红细胞谱系中 APA 的综合图谱存在其他 RNA 加工事件，如替代。 剪接和翻译控制，已知对红细胞生成很重要，并且在某些方面失调 贫血和地中海贫血，表明 APA 改变 3'UTR 的长度/特性也可能影响 该项目旨在通过全面地填补这一知识空白。 使用从收集的 RNA 上进行靶向 3' 末端测序来识别和量化红细胞生成过程中的 APA 红系细胞在整个分化过程中都存在一些基因。 像转录因子 TAL1 (SCL) 和 TCF3 (E2A) 一样，在此过程中会发生 APA 转变。 将通过监测对 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 删除以强制同工型表达）。 该项目将通过分析大量的红细胞生成过程来确定 APA 转变的关键调节因子 K562 红白血病细胞的 RBP 敲低、突变和敲除实验，然后 实验验证初步数据表明剪接因子在骨髓增生异常中常见突变。 诸如 SRSF2 之类的综合征（MDS，一种以红细胞生成无效为特征的疾病）也会影响 综上所述，本提案概述的研究将观察到红细胞分化中的 APA 变化。 深入了解红细胞生成过程中使用的 APA 的新调节机制，从而改变功能 鉴定该过程的分子调节因子，其中一些已经与此相关。 疾病，可能会提出新的治疗途径。