uORF-mediated Translational Control of Cardiac Transcription Factor Expression

uORF介导的心脏转录因子表达的翻译控制

• 批准号: 10659430
• 负责人: Peng Yao
• 金额: $ 50.76万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659430
• 关键词: 5' Untranslated Regions; Animals; Antisense Oligonucleotides; Biochemical; Bioinformatics; Biological; Biological Assay; Biology; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Culture System; Cells; Chemistry; Data Analyses; Databases; Development; Disease Progression; Double-Stranded RNA; Elements; Exhibits; GATA4 gene; Gene Expression Regulation; Genetic; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Homeostasis; Human; Hypertrophy; Initiator Codon; Intervention; Knock-in Mouse; Left; Mediating; Modality; Modeling; Molecular; Morbidity - disease rate; Mus; Open Reading Frames; Output; Pathogenicity; Pathologic; Peptides; Phenotype; Physiology; Protein Biosynthesis; Proteins; RNA; RNA Helicase; Regulation; Repression; Role; Specificity; Stress; Structure; Technology; Therapeutic Intervention; Trans-Activators; Transcript; Translating; Translational Repression; Translations; United States; aorta constriction; design; genome editing; heart function; human disease; human embryonic stem cell; innovation; insight; loss of function; mortality; mouse model; mutant; new therapeutic target; novel; novel therapeutic intervention; pressure; preventive intervention; protein expression; ribosome profiling; synergism; therapeutic RNA; transcription factor

Short peptide-encoding sequences in the 5' untranslated region of messenger ribonucleic acids (mRNA), called upstream open reading frames (uORFs), are widespread in ~50% of human mRNAs. Translating these uORF sequences reduces the protein output of an mRNA main open reading frame (mORF). Our bioinformatic analysis of human and mouse ribosome profiling databases uncovered a group of cardiac mRNA transcripts containing translated uORFs, such as transcription factors, including GATA4. Biochemical analysis suggests that stabilizing the double-stranded RNA (dsRNA) structure downstream of the start codons of these peptide-encoding sequences enhances their translation, thereby inhibiting the translation of mORFs. This translational inhibitory mechanism is mitigated by DEAD-box RNA helicase DDX3X that unwinds dsRNA and inactivates uORF. Genetic depletion of GATA4 uORF activity using CRISPR-Cas9 mediated genomic editing of the start codon in human embryonic stem cells (ESC) provides evidence of uORF-mediated regulation of mORF translation and cardiomyocyte (CM) hypertrophy. In addition, an established CRISPR-Cas9-derived uORF start codon mutant knock-in mouse model shows spontaneous cardiac hypertrophy and will be used to characterize CM hypertrophy at baseline and under stress conditions. Based on our discovered molecular mechanism of DDX3X-regulated, dsRNA-dependent, uORF-mediated translational inhibition of mORF, we have developed two types of antisense oligonucleotides (ASOs) that can either enhance or reduce uORF translation by strengthening or disrupting dsRNA structures. The uORF-enhancing ASO locks the dsRNA structure and activates translation of the uORF, thereby reducing GATA4 mORF protein expression in human CMs. Treatment of mouse cardiomyopathy models with uORF-enhancing ASO reduces GATA4 protein expression, antagonizes cardiac hypertrophy, and restores cardiac function. Based on our findings, we hypothesize that cardiac transcription factor mRNA uORF-dsRNA element acts as a switch for translational control of mORF, regulating cardiac hypertrophy, and can be targeted by ASOs to modulate mORF protein translation and cardiac hypertrophy. We will focus on 3 Specific Aims. Aim 1. Elucidate mRNA structural elements and their interplay with the GATA4 uORF for regulating mORF translation. Aim 2. Determine the biological role of the GATA4 uORF in genetic knock-in mouse models and primary CM cell culture systems. Aim 3. Develop proof-of-concept translation-manipulating ASOs targeting the GATA4 uORF for short-term anti- hypertrophy intervention. These studies will provide novel insights into translational control mechanisms in cardiac biology. This project will promote novel therapeutic approaches (targeting uORF-dsRNA elements) to regulate cardiac hypertrophy. Our mechanism-based design of translation-manipulating ASOs can serve as a proof-of-concept model to apply to different pathogenic mRNA targets.

信使核糖核酸（mRNA）5'非翻译区的短肽编码序列， 称为上游开放阅读框（uORF），广泛存在于约50%的人类mRNA中。翻译 这些uORF序列减少mRNA主要开放阅读框（mORF）的蛋白质输出。我们 对人类和小鼠核糖体分析数据库的生物信息学分析揭示了一组心脏 含有翻译的uORF的mRNA转录物，如转录因子，包括GATA 4。 生化分析表明，稳定双链RNA（dsRNA）结构下游的 这些肽编码序列的起始密码子增强它们的翻译，从而抑制蛋白质的表达。 mORF的翻译。这种翻译抑制机制被DEAD盒RNA解旋酶减轻 DDX 3X解旋dsRNA并灭活uORF。GATA 4 uORF活性的遗传消耗，使用 CRISPR-Cas9介导的人胚胎干细胞（ESC）中起始密码子的基因组编辑提供了 uORF介导的mORF翻译调节和心肌细胞（CM）肥大的证据。在 此外，建立的CRISPR-Cas9衍生的uORF起始密码子突变体敲入小鼠模型显示， 自发性心脏肥大，并将用于表征基线和低于基线时的CM肥大 压力条件。基于我们发现的DDX 3X调节的，dsRNA依赖的， uORF介导的mORF的翻译抑制，我们已经开发了两种类型的反义 可以通过增强或破坏uORF翻译来增强或减少uORF翻译的寡核苷酸（ASO）。 dsRNA结构。增强uORF的阿索锁定双链RNA结构并激活双链RNA的翻译 uORF，从而降低人CM中GATA 4 mORF蛋白的表达。治疗小鼠 具有uORF增强的阿索的心肌病模型降低GATA 4蛋白表达，拮抗 心脏肥大并恢复心脏功能。根据我们的发现，我们假设心脏 转录因子mRNA uORF-dsRNA元件作为mORF翻译控制的开关， 调节心脏肥大，并且可以被ASO靶向以调节mORF蛋白翻译， 心脏肥大我们将专注于三个具体目标。目标1.阐明mRNA结构元件， 它们与GATA 4 uORF相互作用以调节mORF翻译。目标二。确定生物学作用 GATA 4 uORF在遗传敲入小鼠模型和原代CM细胞培养系统中的表达。目标3. 开发靶向GATA 4 uORF的概念验证操纵ASO，用于短期抗肿瘤治疗。 肥大干预这些研究将为翻译控制机制提供新的见解 心脏生物学该项目将促进新的治疗方法（靶向uORF-dsRNA 元素）来调节心脏肥大。我们基于机械原理设计的 ASO可以作为概念验证模型应用于不同的致病性mRNA靶标。