Intron Retention Mechanisms that Regulate Erythroid SF3B1 Gene Expression

调节红细胞 SF3B1 基因表达的内含子保留机制

• 批准号: 9307813
• 负责人: JOHN G CONBOY
• 金额: $ 35.07万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9307813
• 关键词: Achievement; Address; Antisense Oligonucleotides; Automobile Driving; Binding Proteins; Cell Differentiation process; Cell Proliferation; Cells; Data; Defect; Development; Disease; Distal; Distant; Dysmyelopoietic Syndromes; Elements; Engineering; Enhancers; Erythroblasts; Erythroid; Erythropoiesis; Event; Excision; Exhibits; Exons; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hematological Disease; Hematology; Homeostasis; Human; Introns; Investigation; Iron; Lead; Length; Manuscripts; Mediating; Messenger RNA; Metal Ion Binding; Methods; Mission; Mitochondria; Modeling; Molecular; Mutation; Mutation Analysis; NIH Program Announcements; National Institute of Diabetes and Digestive and Kidney Diseases; Oligonucleotides; Orthochromatophilic Erythroblast; Output; Patients; Pattern; Play; Positioning Attribute; Process; Pronormoblasts; Proteins; RNA; RNA Processing; RNA Splicing; Reagent; Regulation; Regulator Genes; Regulatory Element; Reporter; Repression; Research; Role; Site; Specificity; Structure; Syndrome; Techniques; Technology; Testing; Therapeutic; Transcript; Transcription Process; Up-Regulation; base; deletion analysis; design; experience; experimental study; human disease; insight; member; novel; paralogous gene; programs; response; therapy design; transcriptome; treatment strategy

PROJECT SUMMARY/ABSTRACT This SHINE II proposal will address a novel mechanism of gene regulation, intron retention (IR), as it applies to a major splicing regulator / blood disease gene (SF3B1), and an iron homeostasis gene, SLC25A28 (mitoferrin-2). These gene models represent developmentally dynamic and developmentally stable retention events, respectively, that likely are regulated by different mechanisms. Goals of this proposal are to define the mechanism(s), and to obtain proof of principle that this mechanistic information can lead to methods for modulation of retention in a potentially therapeutic manner. IR transcripts, which by definition retain at least one unspliced intron, represent an abundant fraction of many genes' transcriptional output in human erythroblasts: up to 50% in both SF3B1 and SLC25A37 (mitoferrin-1). The latter is a close paralog of SLC25A28 and is very highly expressed in late erythroblasts, but SLC25A28 is more amenable to study due to smaller intron size. IR can theoretically impose major post-transcriptional limits on expression of translatable mRNA during normal development, and by extension mis-regulated could effect quantitative abnormalities of expression in putative `intron retention' diseases. Indeed, aberrant intron retention is a hallmark of myelo- dysplasia syndrome (MDS) in patients with ZRSR2 mutations. Little is known about molecular mechanisms controlling IR. IR in SF3B1 is developmentally dynamic, being up-regulated from 20% to 50% as cells mature from proerythroblasts (lower IR) to orthochromatophilic erythroblasts (higher IR). One model for IR regulation in this gene involves activity of `decoy' or `cryptic' exons in the intron that may interact with the flanking splice sites to block intron excision. In contrast, IR is SLC25A28 (and SLC25A37) is already high in proerythroblasts and remains high throughout erythroblast differentiation. Preliminary data show that an antisense morpholino directed against a distal intron region can alter IR in the endogenous SLC25A28 gene, indicating the presence of an intron splicing enhancer that potentially could act via formation of an RNA bridge. The aim of this SHINE II proposal is to identify cis-regulatory elements in SF3B1 intron 4 and SLC25A28 intron 2 that mediate IR, and to target key regulatory elements with antisense oligonucleotides in an effort to modulate IR efficiency. Regulatory elements will be studied in the context of newly constructed minigene splicing reporters already demonstrated to successfully model IR in transfected cells. Systematic mutation and deletion analysis of the splicing reporter will reveal which regions impact IR, and guide attempts to block these regions with antisense reagents in order to modulate IR. These studies are entirely novel since nothing is known about regulation of IR. The mechanistic information gained here will be relevant to many other genes that also execute IR as part of their expression repertoire, especially other RNA splicing factors that play a huge role in fine tuning transcriptome structure. In the future this data may lead to treatment strategies for emerging diseases characterized by intron retention.

项目摘要/摘要 这项Shine II提案将解决基因调节，内含子保留（IR）的新型机制，因为它适用于 主要的剪接调节剂 /血液疾病基因（SF3B1）和铁稳态基因，SLC25A28 （Mitoferrin-2）。这些基因模型代表了发展动态和发育稳定的保留率 事件分别受不同机制调节的事件。该提议的目标是定义 机制，并获得原理证明，这种机械信息可以导致方法 以潜在的治疗方式调节保留率。 IR成绩单，定义至少保留 一个未填充的内含子代表了人类中许多基因转录输出的大量部分 红细胞：SF3B1和SLC25A37（Mitoferrin-1）中最高50％。后者是 SLC25A28，在较晚的红细胞中非常高度表达，但是SLC25A28更适合于研究 较小的内含子大小。 IR理论上可以对可翻译的表达施加重大的转录后限制 正常发育过程中的mRNA，并且通过扩展错误调节可能会影响定量异常 推定的“内含子保留”疾病中的表达。确实，异常内含子保留是骨髓的标志 ZRSR2突变患者的发育不良综合征（MDS）。关于分子机制知之甚少 控制IR。 SF3B1中的IR在发育上是动态的，随着细胞的成熟，IR从20％上调到50％ 从培养基细胞（下级IR）到正骨成红细胞（较高的IR）。 IR调节的一种模型 在此基因中，内含子中可能与侧翼剪接相互作用的内含子中的“诱饵”或“隐性”外显子的活性 网站以阻止内含子切除。相比之下，IR是SLC25A28（和SLC25A37）的proererythrosplasts很高 并在整个红细胞分化中保持较高。初步数据表明反义吗？ 针对远端内含子区域的针对内源性SLC25A28基因的IR可以改变IR，表明存在 内含子剪接增强子可能通过形成RNA桥而作用。这种光泽的目的 II提案是在SF3B1内含子4和SLC25A28内含子2中识别介导IR的SF3B1内含子中的顺式调节元件，并且 用反义寡核苷酸靶向关键的调节元件，以调节IR效率。 将在新建的小型剪接记者的背景下研究监管元素 证明在转染的细胞中成功模拟IR。系统的突变和缺失分析 剪接记者将揭示哪些区域影响IR，并指导试图用反义阻止这些区域 试剂以调节IR。这些研究完全是新颖的，因为对调节的调节一无所知 ir。此处获得的机械信息将与许多其他也将IR作为一部分执行的基因相关 它们的表达曲目，尤其是其他在微调中起着巨大作用的RNA剪接因子 转录组结构。将来，这些数据可能会导致新兴疾病的治疗策略 以内含子保留为特征。