Analysis of the assembly and functional defect of the spliceosome associated with Retinitis pigmentosa

剪接体是一种高度动态的大分子核糖核蛋白(RNP)机器，通过组装U1、U2、U4、U5和U6小核(Sn)RNP来催化前mRNA剪接。为了处理大量的内含子，SnRNPs的合成和循环必须保持在适当的范围内，以避免它们的短缺，这可能会导致严重的疾病，如视网膜色素变性。然而，维持细胞内SNRNP水平的机制尚不清楚。我们构建了一个与SnRNPs核心成分融合的荧光素酶片段的表达文库，并用它分离了在U5 SnRNP复合体中特异重组荧光素酶活性的PRPF6和U5-40K。我们发现，记者检测到小分子对U5 SnRNP报告蛋白复合体水平的影响。我们的实验将有助于鉴定能够将SnRNP水平控制在适当范围内的小分子。

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear (sn)RNPs. To process large numbers of introns, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage, which would cause a severe disease such as Retinitis pigmentosa. However, the mechanism that maintains cellular snRNP levels is unknown. We constructed an expression library of a luciferase fragment fused to core components of snRNPs and used it to isolate PRPF6 and U5-40K that specifically reconstitute luciferase activity in the U5 snRNP complex. We revealed that the reporter detects the effects of small molecules on the levels of the U5 snRNP-reporter protein complex. Our assay will be useful to identify the small molecule that can control the snRNP level in an appropriate range.