Jean D. Beggs

Yeast Sm-like proteins function in mRNA decapping and decay

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5′-to-3′ decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the ‘Sm’ sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1–Lsm7) also lead to inhibition of mRNA decapping. In addition, the Lsm1–Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery. In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism.

The construction in vitro of transducing derivatives of phage lambda.

SummaryMethods are described for the construction of plaque-forming, transducing derivatives of phage lambda, using appropriate receptor genomes and fragments of DNA generated by the restriction enzymes endo R.EcoRI and endo R.HindIII. The general properties of the transducing derivatives are described and discussed. Plaque-forming phages carrying the E. coli trp, his, cysB, thyA, supD, supE, supF, hsd, tna and lig genes have been isolated.

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

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/U6·U5 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.

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two‐hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two‐hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome‐wide scale. Copyright

A role for Q/N-rich aggregation-prone regions in P-body localization

P-bodies are cytoplasmic foci that are sites of mRNA degradation and translational repression. It is not known what causes the accumulation of RNA-degradation factors in P-bodies, although RNA is required. The yeast Lsm1-7p complex (comprising Lsm1p to Lsm7p) is recruited to P-bodies under certain stress conditions. It is required for efficient decapping and degradation of mRNAs, but not for the assembly of P-bodies. Here we show that the Lsm4p subunit and its asparagine-rich C-terminus are prone to aggregation, and that this tendency to aggregate promotes efficient accumulation of Lsm1-7p in P-bodies. The presence of glutamine- and/or asparagine-rich (Q/N-rich) regions in other P-body components suggests a more general role for aggregation-prone residues in P-body localization and assembly. This is supported by reduced P-body accumulation of Ccr4p, Pop2p and Dhh1p after deletion of these domains, and by the observed aggregation of the Q/N-rich region from Ccr4p.

Splicing-dependent RNA polymerase pausing in yeast.

Summary In eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3′ end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3′ splice sites of constitutively expressed, endogenous yeast genes. We propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.

Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4‐thiouridine at specific sites were used with UV‐crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5′ splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3′ splice site region. Crosslinking of PRP8 to mutant and duplicated 3′ splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5′ exon was established before step 1 and to the 3′ splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.

Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast.

Many protein–protein and protein–nucleic acid interactions have been experimentally characterized, whereas RNA–RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA–RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3′ side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3′ domain and long-range contacts between the 3′ and 5′ domains. CLASH should allow transcriptome-wide analyses of RNA–RNA interactions in many organisms.

Vac14 Controls PtdIns(3,5)P2 Synthesis and Fab1-Dependent Protein Trafficking to the Multivesicular Body

BACKGROUND The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P(2), a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. RESULTS Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P(2), and hyperosmotic shock does not stimulate PtdIns(3,5)P(2) synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. CONCLUSIONS Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P(2) synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P(2), for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.

Lsm proteins and RNA processing1

Sm and Lsm proteins are ubiquitous in eukaryotes and form complexes that interact with RNAs involved in almost every cellular process. My laboratory has studied the Lsm proteins in the yeast Saccharomyces cerevisiae, identifying in the nucleus and cytoplasm distinct complexes that affect pre-mRNA splicing and degradation, small nucleolar RNA, tRNA processing, rRNA processing and mRNA degradation. These activities suggest RNA chaperone-like roles for Lsm proteins, affecting RNA-RNA and/or RNA-protein interactions. This article reviews the properties of the Sm and Lsm proteins and structurally and functionally related proteins in archaea and eubacteria.