title: New insights into the spliceosome by single molecule fluorescence microscopy.
abstract: Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ∼100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA 01605, USA. ahoskins@wisc.edu
authors: Aaron A
authors: AA
authors: Hoskins
authors: Jeff
authors: J
authors: Gelles
authors: Melissa J
authors: MJ
authors: Moore
mesh_terms: D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D005091:Exons; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D007438:Introns; D008856:Microscopy, Fluorescence / Q000379:methods*; D012322:RNA Precursors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012331:RNA, Fungal / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D012322:RNA Precursors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D001710:Biotin; D019809:Streptavidin