Characterization of Sm-like proteins in yeast and their association with U6 snRNA

Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
The EMBO Journal (Impact Factor: 10.75). 08/1999; 18(15):4321-4331. DOI: 10.1093/emboj/18.15.4321
Source: PubMed

ABSTRACT Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis. Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA. Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses. Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA. Analyses of U4/U6 di-snRNPs and U4/U6U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNP in the association–dissociation cycle of spliceosome complexes. Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function. We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing.

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    ABSTRACT: The spliceosome is a large molecular machine that serves to remove the intervening sequences that are present in most eukaryotic pre-mRNAs. At its core are five small nuclear ribonucleoprotein complexes, the U1, U2, U4, U5 and U6 snRNPs, which undergo dynamic rearrangements during splicing. Their reutilization for subsequent rounds of splicing requires reversion to their original configurations, but little is known about this process. Here, we show that ZK863.4/USIP-1 (U Six snRNA-Interacting Protein-1) is a ribonucleotidyl transferase that promotes accumulation of the Caenorhabditis elegans U6 snRNA. Endogenous USIP-1-U6 snRNA complexes lack the Lsm proteins that constitute the protein core of the U6 snRNP, but contain the U6 snRNP recycling factor SART3/B0035.12. Furthermore, co-immunoprecipitation experiments suggest that SART3 but not USIP-1 occurs also in a separate complex containing both the U4 and U6 snRNPs. Based on this evidence, genetic interaction between usip-1 and sart-3, and the apparent dissociation of Lsm proteins from the U6 snRNA during spliceosome activation, we propose that USIP-1 functions upstream of SART3 to promote U6 snRNA recycling. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
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    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. © 2015 Rodgers et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
    RNA 03/2015; DOI:10.1261/rna.047845.114 · 4.62 Impact Factor

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