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

描述（由申请人提供）：大多数人类基因包含至少一个内含子，必须精确拼接以避免在基因表达过程中引入错误。正常剪接模式的破坏可引起或改变人类疾病。剪接体活化是催化前的最后调控步骤，因此必须严格控制以保证剪接的保真度。在剪接体激活过程中，在剪接体中加入U4/U6- u5三snrnp会引发大量RNA重排，尤其是在snRNA U6中。U5解旋酶Brr2解开U4/U6 snRNA双链，从而形成催化必需的结构，如U6内部茎环（ISL）。Prp8 Jab1/MPN结构域调控Brr2的RNA解旋酶活性。剪接体激活的破坏可引起疾病，如色素性视网膜炎（RP）。RP（发病率1/3500）的特点是进行性视网膜变性，最终导致完全失明。U4/U6-U5 tri-snRNP的核心剪接机制成分，特别是PRPC8和SNRNP200的突变，酵母tri-snRNP蛋白Prp8和Brr2的人类同源物，已经与RP有关，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绕U4/U6， Prp8 Jab1/MPN结构域影响Brr2的活性，但U6在激活过程中所经历的动力学和特定构象重排仍然未知，RP等位基因在这些过程中的分子作用也是未知的。先前的研究受到了这样一个事实的阻碍，即大多数剪接研究使用粗剪接提取物，其中剪接体组装中的许多可逆步骤是异步进行的。为了解决这一问题，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，而不需要隔离或同步中间体。本提案中描述的实验首先建立了smFRET检测，以监测纯化的三snRNPs中的U4/U6解旋（目的1），然后使用该系统确定Brr2和Prp8 RP突变体在组装剪接体中是否被错误调节（目的2）。进一步的实验将确定RP突变对剪接效率和保真度的影响。这项工作将

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.