open access

https://hdl.handle.net/2115/56629

A split luciferase-based reporter for detection of a cellular macromolecular complex

英語

Split luciferase

2014-05-01

Elsevier

Analytical Biochemistry

452

1-9

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling Ul, 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. (C) 2014 Elsevier Inc. All rights reserved.

学術雑誌論文

Accepted Manuscript

0003-2697