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.
Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.
Pre-messenger RNA (pre-mRNA) splicing is a central step in gene expression. Lying between transcription and protein synthesis, pre-mRNA splicing removes sequences (introns) that would otherwise disrupt the coding potential of intron-containing transcripts. This process takes place in the nucleus, catalyzed by a large RNA-protein complex called the spliceosome. Prp8p, one of the largest and most highly conserved of nuclear proteins, occupies a central position in the catalytic core of the spliceosome, and has been implicated in several crucial molecular rearrangements that occur there. Recently, Prp8p has also come under the spotlight for its role in the inherited human disease, Retinitis Pigmentosa.Prp8 is unique, having no obvious homology to other proteins; however, using bioinformatical analysis we reveal the presence of a conserved RNA recognition motif (RRM), an MPN/JAB domain and a putative nuclear localization signal (NLS). Here, we review biochemical and genetical data, mostly related to the human and yeast proteins, that describe Prp8's central role within the spliceosome and its molecular interactions during spliceosome formation, as splicing proceeds, and in post-splicing complexes. NOTE ON NOMENCLATUREIn this review, Prp8 and Prp8p represent the protein product of the wild-type PRP8 gene of Saccharomyces cerevisiae, while prp8-1 is an example of a mutant allele of the PRP8 gene. Human Prp8 protein is designated hPrp8, also known in the literature as PRPF8, PRPC8, p220, and 220K. In some places, to avoid confusion, yPrp8 is used to distinguish the yeast (S. cerevisiae) protein from the human form. sPrp8 SPP42 (Schmidt et al. 1999) or sPrp8 cwf6 (McDonald et al. 1999;Ohi et al. 2002) defines the ortholog in Schizosaccharomyces pombe. Confusingly, the cdc28 gene in S. pombe is also referred to as prp8 as a consequence of its role in both pre-mRNA splicing and cell cycle progression, and there is also a temperature-sensitive allele called prp8-1 that causes accumulation of pre-mRNA upon a shift to nonpermissive temperatures (Lundgren et al. 1996;Imamura et al. 1998). Cdc28 prp8 encodes a 112-kDa DEAH-box protein. This protein is not the ortholog of the U5 snRNP protein of S. cerevisiae that is discussed here.In discussions of pre-mRNA-protein cross-links or mutations that affect intron-exon junctions, 5ЈSS+2 refers to the second intronic base from the 5Ј end of the intron, 5ЈSS-2 is the penultimate base of the 5Ј exon, and 3ЈSS-2 is the penultimate base of the intron. PRE-mRNA SPLICINGPre-mRNA splicing involves two trans-esterification reactions within the highly dynamic spliceosome complex. A vast amount of mainly biochemical data led to a consensus view of an ordered pathway of spliceosome assembly that will be described in outline here (for further details, see Kramer 1996;Burge et al. 1999;Brow 2002). The small nuclear RNA-protein (snRNP) complexes, known as U1, U2, U4, U5, and U6 snRNPs, play key roles. U1 is the first snRNP to associate with pre-mRNA, interacting with the 5Ј splice site (5Ј...
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 crosslinking, 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.pre-rRNA | RNA structure | ribosome synthesis | UV cross-linking T he identification of RNA-RNA interactions is essential for detailed understanding of many biological processes. Almost all RNAs must be correctly folded to function, whereas basepairing between different RNA molecules underlies many pathways of RNA metabolism, including pre-mRNA splicing, ribosome synthesis, and the regulation of mRNA stability by microRNAs (miRNAs), among many others. Even for RNAs for which the final structure is known (e.g., rRNA), the folding pathway in precursors is generally unclear. RNA-RNA interactions were previously analyzed by X-ray crystallography, NMR, psoralen cross-linking, and genetics, but all these methods are labor-intensive and typically require prior knowledge of the interacting partners. Because of these technical difficulties, RNA base-pairing is more commonly inferred from a combination of bioinformatic and evolutionary analyses. However, computational methods are applicable only to evolutionarily conserved interactions and provide little information about the physiological context of the interaction.UV cross-linking methods have been developed to map protein interaction sites precisely on RNA molecules, including crosslinking and immunoprecipitation (CLIP) and cross-linking and analysis of cDNAs (CRAC) (1, 2). CRAC analyses have been performed on proteins (Nop1, Nop56, and Nop58) that are associated with all members of the box C/D class of small nucleolar RNAs (snoRNAs). Most box C/D snoRNAs base-pair with the rRNA to select sites of RNA 2′-O-methylation by the methyltransferase fibrillarin (Nop1). In contrast, the U3 snoRNA basepairs to multiple sites on the pre-rRNA. These interactions probably facilitate correct folding of the pre-rRNA and are required for pre-rRNA processing (3, reviewed in refs. 4, 5). PremRNA splicing requires five snRNAs that assemble the complex structure of the spliceosome, with...
SummaryIn 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.
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.
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 Pbodies, 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 Pbodies. 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/Nrich region from Ccr4p. Supplementary material available online at
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