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: 10.43). 08/1999; 18(15):4321-4331. DOI: 10.1093/emboj/18.15.4321
Source: PubMed


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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    • "They are characterized by the presence of the Sm domain (Cooper et al. 1995; Hermann et al. 1995; Seraphin 1995; Salgado-Garrido et al. 1999). The Lsm1-7-Pat1 complex shares six of its seven Lsm subunits (Lsm2 through Lsm7) with the nuclear Lsm2- 8 complex which plays a role in splicing but not in cytoplasmic mRNA decay (Mayes et al. 1999; Bouveret et al. 2000; Tharun et al. 2000; Ingelfinger et al. 2002; Tharun 2009b). Thus, Lsm1 is a key subunit that distinguishes these two complexes. "
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    ABSTRACT: The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.
    RNA 08/2015; 21(8):1469-1479. DOI:10.1261/rna.052209.115 · 4.94 Impact Factor
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    • "The LSm2-8 heptameric complex is located in the nucleus. This complex directly binds and stabilizes the 3′-terminal poly(U) tract of U6 small nuclear RNA, forms the core of the U6 small nuclear ribonucleoproteins (RNPs) and functions in pre-mRNA splicing [12,13]. The Arabidopsis supersensitive to abscisic acid (ABA) and drought 1 (SAD1) gene locus encodes the LSm5 protein and was identified in a genetic screen for components that regulate the expression of stress-responsive genes in our previous work [14]. "
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    ABSTRACT: Sm-like proteins are highly conserved proteins that form the core of the U6 ribonucleoprotein and function in several mRNA metabolism processes, including pre-mRNA splicing. Despite their wide occurrence in all eukaryotes, little is known about the roles of Sm-like proteins in the regulation of splicing. Here, through comprehensive transcriptome analyses, we demonstrate that depletion of the Arabidopsis supersensitive to abscisic acid and drought 1 gene (SAD1), which encodes Sm-like protein 5 (LSm5), promotes an inaccurate selection of splice sites that leads to a genome-wide increase in alternative splicing. In contrast, overexpression of SAD1 strengthens the precision of splice-site recognition and globally inhibits alternative splicing. Further, SAD1 modulates the splicing of stress-responsive genes, particularly under salt-stress conditions. Finally, we find that overexpression of SAD1 in Arabidopsis improves salt tolerance in transgenic plants, which correlates with an increase in splicing accuracy and efficiency for stress-responsive genes. We conclude that SAD1 dynamically controls splicing efficiency and splice-site recognition in Arabidopsis, and propose that this may contribute to SAD1-mediated stress tolerance through the metabolism of transcripts expressed from stress-responsive genes. Our study not only provides novel insights into the function of Sm-like proteins in splicing, but also uncovers new means to improve splicing efficiency and to enhance stress tolerance in a higher eukaryote.
    Genome biology 01/2014; 15(1):R1. DOI:10.1186/gb-2014-15-1-r1 · 10.81 Impact Factor
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    • "We have therefore developed a model of the Lsm1-7-Pat1C complex in several steps. First, sequence alignment shows that each Lsm protein of the Lsm1-7 complex is analogous to one of the seven Sm proteins50,51,52 and thus, the order of Lsm proteins is derived from that of the Sm ring structure53,54. Second, we used the recent crystal structures of Lsm4N and the Lsm5-6-7 complex from Schizosaccharomyces pombe44,55 and human SmB (corresponding to Lsm1) in the spliceosomal U4 snRNP core domain54 to generate homology models of Lsm4, Lsm5, Lsm6, Lsm7 and Lsm1 of S. cerevisiae in the SWISS-MODEL workspace56. "
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    ABSTRACT: The evolutionarily conserved Lsm1-7-Pat1 complex is the most critical activator of mRNA decapping in eukaryotic cells and plays many roles in normal decay, AU-rich element-mediated decay, and miRNA silencing, yet how Pat1 interacts with the Lsm1-7 complex is unknown. Here, we show that Lsm2 and Lsm3 bridge the interaction between the C-terminus of Pat1 (Pat1C) and the Lsm1-7 complex. The Lsm2-3-Pat1C complex and the Lsm1-7-Pat1C complex stimulate decapping in vitro to a similar extent and exhibit similar RNA-binding preference. The crystal structure of the Lsm2-3-Pat1C complex shows that Pat1C binds to Lsm2-3 to form an asymmetric complex with three Pat1C molecules surrounding a heptameric ring formed by Lsm2-3. Structure-based mutagenesis revealed the importance of Lsm2-3-Pat1C interactions in decapping activation in vivo. Based on the structure of Lsm2-3-Pat1C, a model of Lsm1-7-Pat1 complex is constructed and how RNA binds to this complex is discussed.Cell Research advance online publication 19 November 2013; doi:10.1038/cr.2013.152.
    Cell Research 11/2013; 24(2). DOI:10.1038/cr.2013.152 · 12.41 Impact Factor
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