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.

项目总结