Small translated ORFs in the 3'UTR enhance translation in vertebrates

3UTR 中的小翻译 ORF 增强脊椎动物的翻译

• 批准号: 10534126
• 负责人: Ariel Bazzini
• 金额: $ 40.13万
• 依托单位: STOWERS INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10534126
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affect; Amino Acid Sequence; Automobile Driving; Biological; Biological Assay; Biological Process; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Codon Nucleotides; Cytometry; Data; Development; Diagnostic; Disease; Embryo; Flow Cytometry; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Half-Life; Health; High Prevalence; Homologous Gene; Human; Laboratories; Location; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Molecular; Mutation; Mutation Detection; Open Reading Frames; Organism; Outcome; Output; Pathologic; Pathway interactions; Peptides; Phenotype; Post-Transcriptional Regulation; Process; Proteins; Proteomics; RNA; Regulation; Reporter; Research; Ribosomes; Role; Shapes; Stress; Terminator Codon; Therapeutic Intervention; Translating; Translational Regulation; Translations; Untranslated RNA; Untranslated Regions; Vertebrates; Work; Zebrafish; biochemical tools; diagnostic tool; enhancing factor; experimental study; genomic profiles; human disease; innovation; insight; knock-down; novel; overexpression; posttranscriptional; prevent; recruit; ribosome profiling; targeted treatment; tool; transcriptome sequencing; translational engagement

Project Summary The prevailing doctrine that messenger RNAs (mRNAs) in higher organisms encode for a single protein has undergone a dramatic revision in recent years. Ribosome and proteomic profiling have revealed a large number of small translated open reading frames (ORF) within previously described “untranslated regions” (UTRs) and long non-coding RNAs. Indeed, some of the peptides derived from small ORFs have been implicated in various fundamental processes (e.g., development). Translation of small ORFs in the 5’UTR, known as upstream-ORFs (uORFs), has been shown to have a profound regulatory effect on gene regulation, independent of the encoded peptide. Further, translation of uORFs vary under pathologic conditions such as cancer, and mutations affecting uORFs are associated with various human diseases. We and others have also indicated the existence of translated small ORFs in the 3’UTR known as downstream open reading frames (dORFs) in human cells and zebrafish embryos. However, contrary to uORFs, there has been no systematic study of dORF functions, and their relationship to human health and disease remains untested. Further, given their location in the 3’UTR, the molecular mechanism by which dORFs engage the translational machinery remain completely unknown. Our long-term goal is to understand how post-transcriptional regulation (mRNA half-life and translation) shapes gene expression in vertebrates, and its impact on human disease. The central hypothesis of this application is that translation of dORFs regulates gene expression. Our preliminary data strongly indicate that, contrary to uORFs, dORFs strongly enhance translation of the canonical ORF and emerge as an uncharacterized and potent regulatory mechanism across vertebrates. The objectives are to: 1) Identify factors involved in enhancing translation of the main ORF. 2) Dissect the regulatory information driving dORF translation, and 3) Characterize the biological impacts of dORF-mediated regulation. The rationale for the proposed research is to gain a mechanistic understanding of dORF-mediated regulation in order to assess the possible biological importance of dORF dysregulation under stress or disease conditions. This proposal is conceptually innovative as it is based on the exploration of a novel, yet widespread and potent translation regulatory mechanism conserved across vertebrates. Technically, this proposal will combine genomic profiles (RNA-seq, Ribosome profiling); reporter (cytometry); biochemistry tools: RNA pulldowns follow by proteomics, CRISPR-Cas-9 and - 12a (to edit) and our novel Cas13d tool (knock-down in embryos); combining human cell and zebrafish embryos. The outcomes from this project will help understand how dORFs are translated, shape gene expression and generate phenotypes. This novel function of the ribosome adds to the recently emerging regulatory effects of translation on gene expression (e.g. uORF, codon optimality). Understanding dORF biology will provide an entry point and perhaps even a diagnostic tool to associate mutations with human diseases. Identifying the molecular machinery involved in this pathway might provide targets for therapeutic interventions.

项目总结