Post-translational phenotypic profiling through nucleotide barcode sequencing

通过核苷酸条形码测序进行翻译后表型分析

• 批准号: 10649344
• 负责人: NICHOLAS T INGOLIA
• 金额: $ 22.24万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649344
• 关键词: Affect; Amino Acid Sequence; Bar Codes; Binding; Biological; Biological Assay; CRISPR/Cas technology; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; DNA Sequence; DNA biosynthesis; DNA sequencing; Disease; Drug resistance; Enhancers; Ensure; Enzymes; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Goals; Health; High-Throughput Nucleotide Sequencing; Individual; Libraries; Malignant Neoplasms; Maps; Measurement; Measures; Mediating; Molecular; Mutagens; Mutation; Nucleotides; Oncogenic; Outcome; Peptide Hydrolases; Phenotype; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Post-Translational Regulation; Protein Kinase; Proteins; RNA; RNA-Binding Proteins; Regulation; Reporter; Signal Pathway; Signal Transduction; Small RNA; Techniques; Testing; Transcriptional Regulation; Transformed Cell Line; Transgenic Organisms; Translation Process; Translations; Variant; Viral; Work; base editor; deep sequencing; driver mutation; experimental study; genetic variant; innovation; interest; kinase inhibitor; link protein; mRNA sequencing; novel; promoter; protein function; response; transcription factor

ABSTRACT Advances in DNA synthesis and sequencing now enable large-scale experiments that systematically explore how sequence variation affects molecular and cellular function. Transcriptional regulation has proven particularly amenable to massively parallel experiments that have revealed how sequence variation affects the activities of promoters and enhancers as well as transcription factors. These experiments have relied on high-throughput sequencing of the mRNAs produced during transcription as a direct read-out of regulatory activity. This elegant approach cannot be directly extended to study other important modes of biological regulation, however. Post-translational protein modification underlies nearly all cell signaling pathways. Despite its importance, we lack general approaches to assess how sequence variation affects post- translational regulation. We will address this gap with a broadly applicable strategy to link protein modifications, such as phosphorylation or regulated degradation, with a deep sequencing readout. Here we propose to develop this technique and validate it by demonstrating how protein-level regulation is affected by variation in the target protein, the modifying enzyme, and the broader genetic context of the cell.

摘要