The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla. In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA. We have cloned the genes encoding all four protein subunits of endonuclease. Each gene is essential. Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids. This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other Archaea. Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively. The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.

The yeast U4͞U6⅐U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4͞ U6⅐U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4͞U6⅐U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4͞U6⅐U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.Eukaryotic pre-mRNA must undergo extensive processing in the nucleus before it is converted into the mature mRNA species competent for translation into protein. One important processing step is the excision of intervening sequences, termed introns, which are present within the coding sequences. Removal of introns is effected by the pre-mRNA splicing machinery, which is composed of five phylogenetically conserved small nuclear RNAs (snRNAs) assembled into small ribonucleoprotein particles (snRNPs) and several non-snRNP associated proteins (1). Once assembled onto pre-mRNA, the massive RNP termed the spliceosome removes the intronic RNA sequences by two ordered transesterification reactions ligating the two exons together, forming the mature message. The biochemistry of the mammalian spliceosomal snRNPs has been extensively studied, and many of the protein components have been purified and their genes cloned (2-4). The budding yeast Saccharomyces cerevisiae has been very useful in the study of pre-mRNA splicing, as it is amenable to genetic manipulations. The low abundance of premRNA splicing snRNPs, however, has made their isolation technically challenging. Several yeast proteins involved in splicing have been identified by conditional growth mutations that produce a pre-mRNA splicing defect (5-7) from synthetic lethal analysis (8, 9), identification of second-site suppressors (10-13), two-hybrid analysis (14), and, most recently, from the direct biochemical purification of the yeast U1 snRNP (15,16).In yeast, there are an estimated 100-200 copies of each spliceosomal snRNA (17) as compared with 100,000-1,000,000 snRNA copies in mammalian cells (18). This discrepancy in abundance has made the purification of yeast snRNPs much more difficult, especially in the case of the yeast U4͞U6⅐U5 tri-snRNP, which exists in equilibrium with the individual ...

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis.DEXH/D-box proteins comprise a family of RNA helicase and RNA helicase-like proteins that function in almost all aspects of eukaryotic gene expression. Included in these processes is RNA transcription (30, 49), pre-mRNA splicing (reviewed in reference 35), mRNA export (21, 34, 38), translation initiation (40), RNA interference (29, 41), RNA turnover (1), viral replication (31), and ribosome biogenesis (reviewed in references 13 and 44). Among this class of RNA helicases are the DEAD-box proteins and the DEAH-box proteins, which differ by subtle sequence variations in one or more of the motifs that define these proteins (reviewed in reference 42). In addition to an RNA-unwinding activity demonstrated by some of these enzymes, others exhibit an "RNPase" function that serves to displace proteins from or otherwise rearrange ribonucleoproteins (10,20). Although the specific function and mechanism of most DEXH/D-box proteins are unknown, the processes in which many of these enzymes function have been studied in great detail.The eukaryotic pre-mRNA splicing reaction is an ATP-intensive process in which at least eight DEXH/D-box proteins function (35). The genes for all eight Saccharomyces cerevisiae spliceosomal DEXH/D-box proteins are essential, although two have been shown to be dispensable in concert with bypass suppressor mutations (6, 23). DEXH/D-box proteins are required for virtually every known step of the pre-mRNA splicing reaction and are thought to monitor and modify RNA-RNA interactions and RNA-protein interactions in the highly dynamic process.Among the trans-acting spliceosomal RNA helicase-like proteins is Prp43p, which was originally identified in a PCR-based screen for RNA helicase genes in yeast (2, 9). It has been shown to be a bona fide RNA-dependent ATPase, although no RNA-unwinding activity h...

The enzymatic processing of cellular RNA molecules requires selective recognition of unique chemical and topological features. The unusual 2′,5′-phosphodiester linkages in RNA lariats produced by the spliceosome must be hydrolyzed by the intron debranching enzyme (Dbr1) before they can be metabolized or processed into essential cellular factors, such as snoRNA and miRNA. Dbr1 is also involved in the propagation of retrotransposons and retroviruses, although the precise role played by the enzyme in these processes is poorly understood. Here, we report the first structures of Dbr1 alone and in complex with several synthetic RNA compounds that mimic the branchpoint in lariat RNA. The structures, together with functional data on Dbr1 variants, reveal the molecular basis for 2′,5′-phosphodiester recognition and explain why the enzyme lacks activity toward 3′,5′-phosphodiester linkages. The findings illuminate structure/function relationships in a unique enzyme that is central to eukaryotic RNA metabolism and set the stage for the rational design of inhibitors that may represent novel therapeutic agents to treat retroviral infections and neurodegenerative disease.

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