Maita Hiroshi, Tomita Kenji, Ariga Hiroyoshi
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
Anal Biochem. 2014 May 1;452:1-9. doi: 10.1016/j.ab.2014.01.015. Epub 2014 Feb 3.
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 RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.
剪接体是一种高度动态的大分子核糖核蛋白（RNP）机器，它通过组装U1、U2、U4、U5和U6小核核糖核蛋白（snRNP）来催化前体mRNA剪接。为了用有限数量的snRNP处理大量内含子，snRNP的合成和循环必须维持在适当范围内，以避免其短缺。然而，维持细胞snRNP水平的机制尚不清楚。调节细胞snRNP水平的分子可能有助于确定这一机制，但目前尚无此类分子。因此，本研究的目标是基于对snRNP的蛋白质组学分析，利用分裂荧光素酶开发一种snRNP水平报告基因。我们构建了一个与U5 snRNP核心成分融合的荧光素酶片段表达文库，并用它分离出在前体mRNA加工因子6（PRPF6）和小核核糖核蛋白40 kDa（U5-40K），它们能在U5 snRNP复合物中特异性地重建荧光素酶活性。在此我们表明，该报告基因可检测小分子对U5 snRNP报告蛋白复合物水平的影响。当大分子复合物的结构尚未精确确定时，我们的方法为发现靶向该复合物的小分子提供了一种替代检测方法。
