A split luciferase-based reporter for detection of a cellular macromolecular complex.
A split luciferase-based reporter for detection of a cellular macromolecular complex.
复制标题
一种基于分裂荧光素酶的报告基因，用于检测细胞大分子复合物。
DOI：
10.1016/j.ab.2014.01.015
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发表时间：
2014
影响因子：
2.9
通讯作者：
Hiroyoshi Ariga
中科院分区：
文献类型：
--
作者：
Hiroshi Maita;Kenji Tomita;Hiroyoshi Ariga
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RNPs （snRNPs）来催化pre-mRNA剪接。为了用有限数量的snRNPs加工大量内含子，snRNPs的合成和再循环必须保持在适当的范围内，以避免snRNPs的短缺。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定义这一机制，但目前尚不清楚。因此，本研究的目标是基于snRNP的蛋白质组学分析，利用分裂荧光素酶开发snRNP水平的报告基因。我们构建了与U5 snRNP核心组分融合的荧光素酶片段表达文库，并利用它分离出特异性重建U5 snRNP复合体中荧光素酶活性的pre-mRNA加工因子6 （PRPF6）和小核核糖核蛋白40 kDa （U5- 40k）。在这里，我们展示了该报告蛋白检测小分子对U5 snRNP报告蛋白复合物水平的影响。我们的方法提供了另一种检测方法，当复合物的结构不能精确识别时，可以发现靶向大分子复合物的小分子。
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 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.