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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

在信使核糖核酸（mRNA）的5'未翻译区域中的短肽编码序列，在约50％的人类mRNA中，称为上游的开放式阅读框架（UORF）是普遍的。翻译这些UORF序列减少了mRNA主开放阅读框（MORF）的蛋白质输出。我们的人和小鼠核糖体分析数据库的生物信息学分析发现了一组心脏包含翻译UORF的mRNA转录物，例如转录因子，包括GATA4。生化分析表明，稳定下游的双链RNA（DSRNA）结构这些肽编码序列的起始密码子可以增强其翻译，从而抑制 Morfs的翻译。这种翻译抑制作用机制通过死盒RNA解旋酶降低 ddx3x释放dsRNA并使uorf失活。使用GATA4 UORF活性的遗传耗竭人类胚胎干细胞中起始密码子（ESC）的crispr-Cas9介导的基因组编辑（ESC）提供 UORF介导的MORF翻译和心肌细胞（CM）肥大的调节的证据。在此外，已建立的CRISPR-CAS9衍生的UORF起始密码子突变型鼠标模型显示自发性心脏肥大，将用于表征基线和下方CM肥大压力条件。基于我们发现的DDX3X调节的分子机制，DSRNA依赖性， UORF介导的对MORF的翻译抑制作用，我们开发了两种反义寡核苷酸（ASO）可以通过加强或破坏来增强或减少UORF翻译 dsRNA结构。 UORF增强的ASO锁定DSRNA结构并激活翻译 uorf，从而降低了人CMS中的GATA4 MORF蛋白表达。治疗小鼠具有UORF增强ASO的心肌病模型降低了GATA4蛋白的表达，拮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元素充当转化Morf的转换，调节心脏肥大，可以通过ASO来靶向MORF蛋白翻译和心脏肥大。我们将重点关注3个特定目标。目标1。阐明mRNA结构元素和他们与GATA4 UORF进行调节MORF翻译的相互作用。目标2。确定生物学作用遗传敲入小鼠模型和原发性CM细胞培养系统中的GATA4 UORF的。目标3。开发针对GATA4 UORF的概念验证 - 操纵ASOS用于短期抗肥大干预。这些研究将为转化控制机制提供新的见解在心脏生物学中。该项目将促进新颖的治疗方法（针对UORF-DSRNA 元素）调节心脏肥大。我们基于机制的翻译操作设计 ASO可以用作概念验证模型，以应用于不同的致病mRNA靶标。