Identification and characterization of small open reading frames translated during inflammation

炎症期间翻译的小开放阅读框的识别和表征

• 批准号: 10752246
• 负责人: Eric Malekos
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752246
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Amino Acids; Binding; Binding Proteins; Biological Process; Bone Marrow; CRISPR screen; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell Separation; Cell Survival; Characteristics; Chronic; Classification; Code; Communities; Complement; Cytoplasm; DNA; Darkness; Data Set; Detection; Development; Disease; Endotoxins; Eukaryota; Exhibits; Genes; Genetic Transcription; Genome; Goals; Growth; Host Defense; Human; Immunity; Individual; Inflammation; Inflammatory; Inflammatory Response; Interferons; Length; Link; Lipopolysaccharides; Macrophage; Malignant Neoplasms; Mass Spectrum Analysis; Messenger RNA; Modeling; Mus; Myeloid Cells; NF-kappa B; Natural Immunity; Nucleotides; Open Reading Frames; Pathway interactions; Peptides; Phenotype; Play; Polyribosomes; Process; Production; Proteins; Proteome; Publishing; RNA; RNA Splicing; Reporter; Ribosomal RNA; Ribosomes; Role; Saccharomyces cerevisiae; Series; Signal Transduction; Structure; TLR4 gene; Technology; Testing; Therapeutic; Transcript; Translating; Translations; Untranslated RNA; Validation; Work; adenylate; circular RNA; cytokine; experimental study; genome annotation; heuristics; interest; new technology; next generation sequencing; novel; pathogen; peptide I; polypeptide; screening; success; therapeutic target; transcriptome

PROJECT SUMMARY Next generation sequencing technologies have greatly expanded the size of the known transcriptome. Many newly discovered transcripts are classified as long noncoding RNAs (lncRNAs) which are assumed to influence phenotype through sequence and structure and not via translated protein products, despite the vast majority of them harboring short open reading frames (sORFs). Recent advances have demonstrated that the noncoding designation is incorrect in many cases and that sORF-encoded peptides (SEPs also called micropeptides) translated from these transcripts are important contributors to diverse biological processes including inflammation and cell viability. An appropriate inflammatory response is critical for host defense against pathogens, but chronic inflammation is associated with many diseases. Macrophages play a significant role in both initiating and resolving inflammation and understanding their part in this process is scientifically and practically important. One long studied - yet not fully understood - model of macrophage proinflammatory polarization involves lipopolysaccharide (LPS) activation of toll-like receptor 4 (TLR4). Following detection of LPS, a signaling cascade initiates leading to the translocation of transcription factor NFkB to the nucleus. This is followed by increased expression of established inflammatory cytokine and interferon genes. However, this also results in changes in expression of many unstudied lncRNAs. In addition to changes in transcription, changes in translation also follow inflammatory stimulation and these alterations have been observed to increase translation of “noncoding” regions in some cases. Indeed our lab and others have observed dramatic changes in associations between lncRNAs and polysomes following LPS stimulation in mouse macrophages. Therefore, the central hypothesis of this proposal is that translation of lncRNAs produce SEPs that play important roles in the TLR4-NFkB inflammatory response and in macrophage viability. To test this hypothesis, I present a strategy for screening lncRNA sORFs with evidence of coding potential in mouse macrophages. The screen will make use of macrophage cell lines with a NFkB-GFP reporter and CRISPR- Cas9 casssette. Secondly, I propose a biomolecular pipeline for mechanistically characterizing a selection of novel SEPs. This work has the potential to identify many novel SEPs that are important for regulating the inflammatory response. This would further our understanding of a model inflammatory pathway and could help identify novel peptides with therapeutic potential or as therapeutic targets.

项目概要 下一代测序技术极大地扩展了已知转录组​​的大小。 新发现的转录本被归类为长非编码 RNA (lncRNA)，它们被认为会影响 通过序列和结构而不是通过翻译的蛋白质产物来确定表型，尽管绝大多数 它们含有短的开放阅读框（sORF），最近的进展表明非编码。 在许多情况下，命名是不正确的，sORF 编码的肽（SEP 也称为微肽） 从这些转录本翻译而来的转录物是多种生物过程的重要贡献者，包括 适当的炎症反应对于宿主防御至关重要。 病原体，但慢性炎症与许多疾病有关。 引发和解决炎症并了解其在这一过程中的作用是科学且可靠的 一种长期研究但实际上尚未完全理解的巨噬细胞促炎模型。 极化涉及在检测到 Toll 样受体 4 (TLR4) 后脂多糖 (LPS) 的激活。 LPS 是一种信号级联启动，导致转录因子 NFkB 易位至细胞核。 随后是已建立的炎症细胞因子和干扰素基因的表达增加。 除了转录的变化之外，还会导致许多未经研究的 lncRNA 的表达发生变化。 翻译的变化也会随着炎症刺激而发生，并且这些变化已被观察到 事实上，在某些情况下，我们的实验室和其他人已经观察到了戏剧性的“非编码”区域的翻译。 小鼠巨噬细胞受到 LPS 刺激后，lncRNA 与多聚核糖体之间的关联发生变化。 因此，该提案的中心假设是lncRNA的翻译产生的SEP 在 TLR4-NFkB 炎症反应和巨噬细胞活力中发挥重要作用。 假设，我提出了一种筛选 lncRNA sORF 的策略，并提供了小鼠编码潜力的证据 筛选将利用带有 NFkB-GFP 报告基因和 CRISPR-的巨噬细胞系。 其次，我提出了一种生物分子管道，用于机械表征一系列物质。 这项工作有可能发现许多对监管非常重要的新颖标准必要专利。 这将进一步加深我们对模型炎症途径的理解，并可能有所帮助。 识别具有治疗潜力或作为治疗靶点的新型肽。