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.

项目概要/摘要 SHINE II 提案将解决基因调控的新机制，即内含子保留（IR），因为它适用于 主要剪接调节因子/血液疾病基因 (SF3B1) 和铁稳态基因 SLC25A28 （线粒体铁蛋白-2）。这些基因模型代表了发育动态和发育稳定的保留 分别可能由不同机制调节的事件。该提案的目标是定义 机制，并获得原理证明，证明该机制信息可以导致方法 以潜在的治疗方式调节保留。 IR 成绩单，根据定义至少保留 一个未剪接的内含子，代表人类许多基因转录输出的丰富部分 成红细胞：SF3B1 和 SLC25A37（线粒体铁蛋白-1）中高达 50%。后者是一个密切的旁系同源 SLC25A28 在晚期成红细胞中表达非常高，但 SLC25A28 更适合研究，因为 内含子尺寸更小。理论上，IR 可以对可翻译的表达施加主要的转录后限制。 正常发育过程中的 mRNA 以及错误调节可能会影响 mRNA 的数量异常 在假定的“内含子保留”疾病中的表达。事实上，异常的内含子保留是骨髓- ZRSR2 突变患者的不典型增生综合征 (MDS)。对分子机制知之甚少 控制IR。 SF3B1 中的 IR 具有发育动态性，随着细胞成熟，IR 会从 20% 上调至 50% 从原红细胞（较低 IR）到正染色质红细胞（较高 IR）。 IR 监管的一种模型 该基因涉及内含子中“诱饵”或“神秘”外显子的活性，可能与侧翼剪接相互作用 阻止内含子切除的位点。相比之下，SLC25A28（和SLC25A37）的IR在原红细胞中已经很高 并在成红细胞分化过程中保持高水平。初步数据表明反义吗啉代 针对远端内含子区域可以改变内源性 SLC25A28 基因中的 IR，表明存在 内含子剪接增强子可能通过形成 RNA 桥发挥作用。此次SHINE的目的 II 建议是鉴定 SF3B1 内含子 4 和 SLC25A28 内含子 2 中介导 IR 的顺式调控元件，以及 利用反义寡核苷酸靶向关键调控元件，以调节 IR 效率。 将在新构建的小基因剪接报告基因的背景下研究调控元件 证明可以成功地模拟转染细胞中的 IR。系统突变和缺失分析 剪接记者将揭示哪些区域影响IR，并指导尝试用反义阻断这些区域 试剂以调节IR。这些研究完全是新颖的，因为对监管的了解一无所知 红外。这里获得的机制信息将与许多其他也执行 IR 作为一部分的基因相关 它们的表达库，尤其是在微调中发挥巨大作用的其他 RNA 剪接因子 转录组结构。未来这些数据可能会导致新出现疾病的治疗策略 其特征是内含子保留。