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Defining the regulatory roles of alternative ribosome initiation and novel peptides

定义替代核糖体起始和新型肽的调节作用

基本信息

批准号：
10299750
负责人：
Jin Chen
金额：
$ 24.9万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-09 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10299750
关键词：
Address Biochemical Biological Biological Models Biology Biophysics CRISPR screen CRISPR/Cas technology Cell Line Cell Survival Cell physiology Cells Cellular Stress Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Complement Data Development Disease Event FRAP1 gene Gene Expression Regulation Genes Genetic Genome Genomic approach Goals Health Transition Human Human Biology Human Genome Project Initiator Codon Knock-in Knock-out Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Mentors Microscopy Modeling Monitor Noise Normal Cell Oncogenes Oncogenic Open Reading Frames Organoids Pathway interactions Peptides Phase Phenotype Protein Biosynthesis Proteins Proteome RNA RNA-Binding Proteins Regulation Regulatory Pathway Reporter Research Personnel Ribosomal RNA Ribosomes Role Signal Pathway Site Stress System Techniques Technology Testing Therapeutic Time Training Trans-Activators Translating Translations Untranslated RNA Variant base cell transformation deep sequencing design experience experimental study follow-up functional genomics gene synthesis genome annotation genome-wide insight knock-down neoplastic cell novel programs protein expression response ribosome profiling single molecule skills targeted treatment tool transcriptome transcriptome sequencing translation factor tumor growth tumorigenesis

项目摘要

PROJECT SUMMARY/ABSTRACT More than 15 years after the completion of the Human Genome Project, our understanding of the “annotated” genome is still incomplete. Alternative ribosome initiation sites that are not part of the “annotated genome” encode novel open reading frames (ORFs) that are either variants of annotated proteins, distinct ORFs upstream or downstream of annotated proteins, or even ORFs on long non-coding RNAs. These novel ORFs are emerging as a translational control mechanism to rapidly reprogram specific genes and protein synthesis networks, especially during stress and cell-state transformations such as tumorigenesis. Furthermore, these new ORFs encode putative peptides that remain uncharacterized. Thus, understanding the translational control mechanisms, as well as the regulatory functions of the encoded peptides, could reveal fundamental biology and targets for therapeutics. Existing studies of alternative initiation or alternative ORF-encoded peptides have taken an ad hoc approach, owing to a lack of tools to profile them at genome-wide scale. Our goal is to develop and apply deep sequencing and high-throughput, CRISPR-based methods to map the functional roles of these nonconventional translation transcriptome-wide, using tumorigenesis as a model system. Functional genomic approaches are uniquely poised to address the deficit in our understanding of functional alternative ORFs. In this proposal we will aim to characterize the global utilization of nonconventional initiation sites during tumorigenesis using ribosome profiling and RNA-seq at various time points (Aim 1). This will define novel ORFs that are actively translated, and how the translation of these ORFs are regulated to promote expression of oncogenic genes and peptides. Then, we will use CRISPR screens to identify ORFs necessary for tumor growth, and define the functions of the novel peptides by characterizing localization, physical interactions, and genetic interactions (Aim 2). Finally, we will mechanistically interrogate alternative start site usage to investigate how translation is tuned during cell-state changes (Aim 3). Overall, the results from the proposal will address long- standing questions about translational control, and reveal the regulatory roles of novel proteins. The combination of mentored support, skills, and data obtained in the K99 phase will provide Dr. Chen a springboard to achieving independence as an investigator in the R00 phase and beyond. The results of our studies will provide new insights into fundamental aspects of translational control, and will define new paradigms relevant to biology and disease.

项目总结/文摘