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.

项目摘要 下一代测序技术极大地扩展了已知转录组的大小。许多 新发现的转录本被归类为长链非编码RNA（lncRNA）， 表型通过序列和结构，而不是通过翻译的蛋白质产物，尽管绝大多数 它们具有短的开放阅读框架（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） 从这些转录本中翻译出来的蛋白质是多种生物过程的重要贡献者， 炎症和细胞活力。适当的炎症反应对于宿主防御 病原体，但慢性炎症与许多疾病有关。宏观经济在以下方面发挥着重要作用： 引发和解决炎症以及了解它们在这一过程中的作用是科学的， 实际上很重要一个长期研究但尚未完全理解的巨噬细胞促炎模型 极化涉及Toll样受体4（TLR 4）的脂多糖（LPS）活化。检测后 LPS是一种信号级联反应，其启动导致转录因子NF κ B易位至细胞核。这 随后是已建立的炎性细胞因子和干扰素基因的表达增加。但这 也导致许多未研究的lncRNA表达的变化。除了转录的变化， 翻译的变化也跟随炎症刺激，并且已经观察到这些变化， 在某些情况下增加“非编码”区域的翻译。事实上，我们的实验室和其他人已经观察到戏剧性的 LPS刺激小鼠巨噬细胞后lncRNA和多聚体之间相关性的变化。 因此，该提议的中心假设是lncRNA的翻译产生SEP， 在TLR 4-NFkB炎症反应和巨噬细胞活力中起重要作用。为了验证这一 假设，我提出了一个策略，筛选lncRNAsORF的证据编码潜力的小鼠 巨噬细胞该筛选将使用具有NF kB-GFP报告基因和CRISPR-1的巨噬细胞系。 九号录音带。其次，我提出了一种生物分子管道，用于机械地表征一系列生物分子。 新型SEP。这项工作有可能确定许多新的SEPs，这些SEPs对调节细胞凋亡非常重要。 炎症反应。这将进一步加深我们对炎症通路模型的理解， 鉴定具有治疗潜力或作为治疗靶点的新肽。