Spliceosome Activation at the Single Molecule Level: Insight from Disease Alleles

单分子水平的剪接体激活：来自疾病等位基因的见解

• 批准号: 8783130
• 负责人: Megan Mayerle
• 金额: $ 5.33万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783130
• 关键词: ATP phosphohydrolase; Address; Affect; Alleles; Biochemical Genetics; Biological Assay; Blindness; C-terminal; Catalysis; DNA Sequence Rearrangement; Disease; Ensure; Eukaryota; Excision; Gene Expression; Genes; Goals; Human; Incidence; Inherited; Introns; Investigation; Kinetics; Label; Link; Malignant Neoplasms; Messenger RNA; Metabolic Diseases; Methods; Molecular; Monitor; Mutation; Orthologous Gene; Pattern; Process; Proteins; RNA; RNA Helicase; RNA Splicing; Regulation; Reporter; Research; Retina; Retinal Degeneration; Retinitis Pigmentosa; Site; Small Nuclear RNA; Small Nuclear Ribonucleoproteins; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Testing; U6 small nuclear RNA; Work; Yeasts; disorder prevention; fluorophore; helicase; human disease; in vivo; insight; mRNA Precursor; mutant; premature; protein complex; public health relevance; research study; single molecule; single-molecule FRET; stem

DESCRIPTION (provided by applicant): The majority of human genes contain at least one intron that must be precisely spliced to avoid introducing errors during gene expression. Disruption of normal splicing patterns can cause or modify human disease. Spliceosome activation is the final regulatory step before catalysis, and therefore must be strictly controlledto ensure splicing fidelity. Addition of the U4/U6-U5 tri-snRNP to the spliceosome during spliceosome activation instigates a large number of RNA:RNA rearrangements, especially in snRNA U6. U5 helicase Brr2 unwinds the U4/U6 snRNA duplex allowing catalytically necessary structures like the U6 internal stem-loop (ISL) to form. The Prp8 Jab1/MPN domain modulates the RNA helicase activities of Brr2. Disruption of spliceosome activation can cause disease, as occurs in Retinitis pigmentosa (RP). RP (incidence 1/3500) is characterized by progressive retinal degeneration that ultimately proceeds to total blindness. Core splicing machinery components of the U4/U6-U5 tri-snRNP, particularly mutations in PRPC8 and SNRNP200, the human orthologs of yeast tri-snRNP proteins Prp8 and Brr2, have been linked to RP however how these mutant alleles affect splicing to cause disease is unknown. In particular, while it is known that Brr2 unwinds U4/U6 during spliceosome activation and that the Prp8 Jab1/MPN domain affects Brr2 activity, the kinetics and specific conformational rearrangements undergone by U6 during activation remain unknown, as are the molecular effects of RP alleles on these processes. Previous investigations were hindered by the fact that most splicing studies use crude splicing extract, wherein the many reversible steps in spliceosome assembly proceed asynchronously. To address this issue single-molecule FRET (smFRET) approaches to monitor splicing have been developed. smFRET provides a method to monitor dynam- ics and conformational rearrangements without the need to isolate or synchronize intermediates. The experi- ments described within this proposal first establish a smFRET assay to monitor U4/U6 unwinding in purified tri- snRNPs (Aim 1) and then use this system to determine if Brr2 and Prp8 RP mutants are misregulated in the assembled spliceosome (Aim 2). Further experiments will determine how splicing efficiency and fidelity is af- fected by RP mutants. Together this work will provide essential fundamental information on spliceosome activa- tion and how aberrant activation causes disease.

描述(申请人提供)：大多数人类基因包含至少一个内含子，必须精确剪接，以避免在基因表达过程中引入错误。正常剪接模式的中断可能会导致或改变人类疾病。剪接体激活是催化前的最后一个调控步骤，因此必须严格控制以确保剪接的保真度。在剪接体激活过程中，将U4/U6-U5 Tri-SnRNP添加到剪接体中会引发大量的RNA：RNA重排，特别是在SnRNAU6中。U5解旋酶Brr2解开U4/U6 SnRNA双链，使U6内部茎环(ISL)等催化必需结构得以形成。Prp8Jab1/MPN结构域调控Brr2的RNA解旋酶活性。剪接体激活的中断会导致疾病，就像视网膜色素变性(RP)一样。RP(发病率1/3500)以进行性视网膜变性为特征，最终进展为完全失明。U4/U6-U5 Tri-SnRNP的核心剪接机制组件，特别是酵母Tri-SnRNP蛋白Prp8和Brr2的人类同源基因PRPC8和SNRNP200的突变，已与RP联系在一起，但这些突变等位基因如何影响剪接导致疾病尚不清楚。特别是，虽然已知Brr2在剪接体激活过程中解开U4/U6，Prp8Jab1/MPN结构域影响Brr2活性，但U6在激活过程中经历的动力学和特定构象重排仍不清楚，RP等位基因在这些过程中的分子作用也是未知的。以前的研究受到这样一个事实的阻碍，即大多数剪接研究使用粗剪接物，其中剪接体组装中的许多可逆步骤是异步进行的。为了解决这个问题，人们开发了单分子FRET(SmFRET)方法来监测剪接。SmFRET提供了一种监测动力学和构象重排的方法，而不需要分离或同步中间体。本建议中描述的实验首先建立了一种smFRET方法来监测纯化的tri-SnRNPs中U4/U6的解离(目标1)，然后使用该系统来确定Brr2和Prp8 RP突变体是否在组装的剪接体中被错误调控(目标2)。进一步的实验将确定RP突变体如何影响剪接效率和保真度。这项工作将共同努力 提供有关剪接体激活和异常激活如何导致疾病的基本信息。