title: Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull.
abstract: Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments.
authors: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors: Margaret L
authors: ML
authors: Rodgers
authors: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors: Joshua
authors: J
authors: Paulson
authors: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors: Aaron A
authors: AA
authors: Hoskins
mesh_terms: D002457:Cell Extracts / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D012313:RNA / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000302:isolation & purification; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000032:analysis* / Q000302:isolation & purification*; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002457:Cell Extracts; C401305:Prp24 protein, S cerevisiae; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405483:U1 small nuclear RNA; D012313:RNA
keywords: fluorescence; microscopy; single-molecule; snRNP; spliceosome; splicing