Analysis of the Role of Post-Transcriptional Regulation by the Poly(C) Binding Proteins in Pancreatic Beta Cell Homeostasis

聚 (C) 结合蛋白转录后调节在胰腺 β 细胞稳态中的作用分析

• 批准号: 10020171
• 负责人: Matthew Haemmerle
• 金额: $ 3.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10020171
• 关键词: Address; Adoption; Affect; Affinity Chromatography; Alleles; Beta Cell; Binding; Binding Proteins; Binding Sites; Blindness; Blood Glucose; Breeding; CRISPR/Cas technology; Cell Line; Cell Maintenance; Cell Survival; Cell physiology; Cells; Chronic; Data; Endocrine; Exposure to; Failure; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Glucagon; Glucose; Goals; Homeostasis; Human; Hyperglycemia; Immunoprecipitation; Impairment; Individual; Insulin; Insulin Resistance; Islets of Langerhans; Kidney Failure; Laboratories; Lead; Lower Extremity; Mediating; Messenger RNA; Metabolic; Metabolism; Morphology; Mus; Non-Insulin-Dependent Diabetes Mellitus; Pathogenesis; Phenotype; Physiological; Play; Poly C; Polyadenylation; Post-Transcriptional Regulation; Protein Family; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Ribosomes; Role; Severities; Shapes; Site; Somatostatin; Structure of beta Cell of islet; Testing; Therapeutic; Time; Tissues; Transcript; Translating; Workload; base; blood glucose regulation; cohort; crosslink; crosslinking and immunoprecipitation sequencing; experimental study; glucose tolerance; impaired glucose tolerance; in vivo; insight; insulin secretion; insulin tolerance; islet; limb amputation; novel; preservation; prevent; response; therapeutic development; transcription factor; transcriptome

Project Summary β cell dysfunction and β cell failure play central roles in the pathogenesis of Type 2 Diabetes Mellitus (T2DM). Increasing evidence has suggested that post-transcriptional regulation of gene expression is essential for β cell function and survival. However, the underlying mechanisms governing how the post-transcriptional landscape is shaped to promote β homeostasis remains largely unexplored. A growing body of evidence has implicated RNA binding proteins (RBPs) in influencing the metabolism of mRNAs that encode products essential for β cell survival. Despite these findings, few RBPs have been examined in understanding how the post-transcriptional milieu is shaped to maintain β cell homeostasis. Based on a Translating Ribosomal Affinity Purification (TRAP) screen in our laboratory using the Min6 β cell line, the poly(C) binding protein family of RNA binding proteins, specifically PCBP1 and PCBP1, have been raised as potential post-transcriptional regulators of β cell identity and survival genes. Thus, the goal of this proposal is to elucidate the mechanistic and physiological importance of post-transcriptional regulation by PCBP1 and PCBP2 in β cells. In the first aim of this proposal, the in vivo roles exerted by PCBP1 and PCBP2 will be examined by crossing mice harboring conditional alleles for Pcbp1 and Pcbp2 with a β cell specific Cre deleter strain. Morphological and functional analyses will be performed with β cells that have individual and combined Pcbp1/2 deficiency. Further, the inclusion of a lineage trace allele will allow us to determine the fate of Pcbp1/2 deficient β cells. The second part of this proposal will examine how glucose shapes the post-transcriptional landscape of β cells. β cells are highly regulated by glucose to allow for tight regulation of blood glucose homeostasis. Interestingly, preliminary demonstrate that Pcbp2 expression substantially increases in murine and human islets exposed to high glucose, pointing to a role in PCBP2 post-transcriptionally mediating the effects of glucose in the β cell. To determine the influence of glucose on the post-transcriptional landscape and the role of PCBP2 in this response, TRAP will be used in wild-type and Pcbp2-deficient β cells exposed to low and high glucose. TRAP has the distinct advantage of determining ribosomal occupancy, or translational efficiency, specifically in β cells within isolated pancreatic islets, which are a heterogenous tissue type. At the same time, analysis of total RNA from isolated β cells will reveal to what extent PCBP2 impacts other aspects of post-transcriptional gene regulation affected by elevated glucose. Additionally, Cross-Linking and Immunoprecipitation Sequencing (CLIP-Seq) will be performed in wild-type and Pcbp2-deficient β cells to identify PCBP2 binding sites transcriptome-wide to determine whether PCBP2 binds to transcripts it functionally impacts. Altogether, these experiments will establish a paradigm for PCBP1 and PCBP2 in preserving β cell homeostasis and will provide mechanistic insight into post- transcriptional regulation of β cell gene expression which could be leveraged in efforts to prevent T2DM.

项目摘要 β细胞功能障碍和β细胞衰竭在2型糖尿病（T2 DM）的发病机制中起着核心作用。 越来越多的证据表明，基因表达的转录后调节对于β细胞至关重要 功能和生存。然而，控制转录后景观的潜在机制 促进β稳态的形状在很大程度上尚未探索。越来越多的证据表明 RNA结合蛋白（RBP）对β细胞必需产物mRNA代谢的影响 生存尽管有这些发现，很少有限制性商业惯例已被审查，了解如何转录后 环境被成形以维持β细胞稳态。翻译核糖体亲和纯化（TRAP） 在我们的实验室中使用Min 6 β细胞系，RNA结合蛋白的poly（C）结合蛋白家族， 特别是PCBP 1和PCBP 1，已经被提出作为β细胞身份的潜在转录后调节因子 生存基因。因此，本建议的目标是阐明机械和生理的重要性， PCBP 1和PCBP 2在β细胞中的转录后调节。在该提议的第一个目的中，体内 PCBP 1和PCBP 2发挥的作用将通过携带Pcbp 1条件等位基因的小鼠杂交来检查 和Pcbp 2与β细胞特异性Cre删除菌株。将进行形态和功能分析 β细胞具有单个和组合的Pcbp 1/2缺陷。此外，包括血统痕迹 等位基因将使我们能够确定Pcbp 1/2缺陷β细胞的命运。本提案的第二部分将 研究葡萄糖如何塑造β细胞的转录后景观。β细胞受到以下因素的高度调节： 葡萄糖，以允许严格调节血糖稳态。有趣的是，初步研究表明， pcbp 2的表达在暴露于高糖的鼠和人胰岛中显著增加，这表明pcbp 2在高血糖中的作用。 在PCBP 2转录后介导葡萄糖在β细胞中的作用中的作用。确定…的影响 葡萄糖对转录后景观的影响以及PCBP 2在这种反应中的作用，TRAP将用于 野生型和Pcbp 2缺陷β细胞暴露于低和高葡萄糖。TRAP具有以下明显优势： 确定核糖体占有率或翻译效率，特别是在分离的胰腺内的β细胞中， 胰岛是一种异质组织类型。同时，从分离的β细胞中分析总RNA， 揭示PCBP 2在多大程度上影响受升高的PCBP 2影响的转录后基因调控的其他方面。 葡萄糖此外，还将在2015年进行交联和免疫沉淀测序（CLIP-Seq）。 野生型和Pcbp 2缺陷型β细胞，以鉴定全转录组的PCBP 2结合位点，以确定是否 PCBP 2与其功能性影响的转录物结合。总之，这些实验将建立一个范例， PCBP 1和PCBP 2在维持β细胞稳态中的作用，并将为β细胞凋亡后的机制提供深入的了解。 β细胞基因表达的转录调节，这可以用于预防T2 DM。