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.

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

“…Given these advantages, SiMPull may prove to be a valuable tool for plant researchers conducting interaction studies with endogenous proteins as well as those limited by antibodies with low affinities. In addition, unlike most assays, SiMPull is not limited solely to protein-protein interactions but can also quantitatively interrogate interactions between proteins and compounds, such as nucleic acids, small molecule ligands, and lipids (Jain et al, 2011(Jain et al, , 2014Krishnan et al, 2013;Hwang et al, 2014aHwang et al, , 2014bRodgers et al, 2015;Arauz et al, 2016).…”

“…Colocalization frequency is known to be strongly influenced by fluorophore maturation probabilities; for instance, as ;80% of YFP and 40% of mCherry molecules are fluorescently active in mammalian systems, measurements of colocalization never reach 100% (reviewed in Aggarwal and Ha, 2014;Isacoff, 2007, 2008;Jain et al, 2014;Rodgers et al, 2015). Our findings thus hint at differences in fluorophore maturation probabilities between animal and plant systems, prompting us to experimentally determine these properties for YFP and mCherry in infiltrated N. benthamiana leaves.…”

“…As SiMPull is compatible with FRET (Jain et al, 2011;Krishnan et al, 2013;Hwang et al, 2014a), dynamic interactions between immunoprecipitated proteins and their binding partners can easily be tested. Finally, SiMPull is not limited solely to protein-protein interactions but provides single-molecule resolution data on interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids (Jain et al, 2011(Jain et al, , 2014Krishnan et al, 2013;Rodgers et al, 2015;Arauz et al, 2016).…”

Lights, camera, action! Capturing the spliceosome and pre‐mRNA splicing with single‐molecule fluorescence microscopy

“…All of the above experiments rely on using CoSMoS to study splicing factors assembling on surface‐tethered pre‐mRNAs. To study interactions between splicing factors in the absence of spliceosome assembly, Rodgers et al recently combined CoSMoS with Single‐Molecule Pull‐down (SiMPull) . SiMPull methods enable capture and single‐molecule characterization of cellular complexes from lysates by pull‐down of FP‐tagged molecules with antibodies directly onto glass microscope slides.…”

“…Rodgers et al optimized the SiMPull approach by developing SNAP‐SiMPull to facilitate labeling and isolation of complexes from yeast extract. SNAP‐SiMPull relies on bright, photostable organic fluorophores—an advantage compared to FPs which sometimes fail to mature and fluoresce when expressed in yeast . To demonstrate the usefulness of this approach, Rodgers et al used SNAP‐SiMPull to label and isolate the branchpoint bridging protein (BBP) and study its interactions with RNAs as well as to demonstrate that an intact yeast U6 snRNA is required for colocalization of the Lsm and Prp24 proteins.…”