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.

项目摘要 成熟Messenger RNA（mRNA）的3'未翻译区域（3'UTR）是停止之间的序列 编码序列和poly（a）尾巴的密码子。重要的是，3'End处理机械的位置 将poly（a）尾巴添加到前mRNA并不是不变的，而是以受控方式变化以生成3'UTR 同一基因的mRNA之间的同工型多样性。该过程称为替代聚腺苷酸化 （APA）。尽管通过APA产生的3'UTR同工型不会改变 蛋白质，它们通过添加或去除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）。有针对性的3英寸 测序技术已显示APA是宽度的，并且在时间之间和特定的时机之间进行了调节 疾病环境。尽管APA的流行率，但对RBP驱动此过程的监管理解 仍然有限。发现特定造血谱系的细胞显示出普遍的APA，但没有 存在红细胞谱系中APA的综合图。其他RNA处理事件，例如替代事件 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改变了3'UTR的长度/身份也可能会影响 健康和疾病的红细胞生物学。该项目旨在通过全面填补这一知识差距 使用靶向的3'端测序在红细胞生成期间识别和量化APA，从 在整个分化过程中，红细胞细胞。初步数据表明，几种基因对促红细胞生成至关重要， 像转录因子TAL1（SCL）和TCF3（E2A）一样，在此过程中会发生APA变化。功能 不同3'UTR同工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 （荧光素酶测定）和分化效率（CRISPR缺失以迫使同工型表达）。最后，这个 项目将通过分析大量纲要来确定APA跨红细胞转移的关键调节剂 来自K562红血球细胞的RBP敲低，突变和敲除实验，然后 实验验证。初步数据表明骨髓增生的剪接因子通常突变 综合征（MDS，一种疾病，部分由无效的红细胞生成），例如SRSF2，也会影响 在红系分化中观察到的APA偏移。综上所述，该提议概述的研究将 提供有关在功能上改变的红细胞生成期间使用的APA新型调节机制的见解 关键基因。识别该过程的分子调节因子，其中一些已经与 疾病可能表明新型治疗途径。