title: A split luciferase-based reporter for detection of a cellular macromolecular complex.
abstract: 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.
authors: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan. Electronic address: maimai@pharm.hokudai.ac.jp.
authors: Hiroshi
authors: H
authors: Maita
authors: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
authors: Kenji
authors: K
authors: Tomita
authors: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan.
authors: Hiroyoshi
authors: H
authors: Ariga
mesh_terms: D000818:Animals; D001483:Base Sequence; D017930:Genes, Reporter / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D051379:Mice; D040901:Proteomics; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C408605:PRPF6 protein, human; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D014157:Transcription Factors; D008156:Luciferases
keywords: Spliceosome; Split luciferase; snRNP