Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition.

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation.

U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute to PRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutant prp38-1. This suppression is specific in that enhanced SPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of other prp mutants. Since SPP381 does not suppress a prp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. Yeast SPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein. A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p-Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing. Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing. Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway.

Core snRNP proteins bind snRNA through the conserved Sm site, PuA(U)n>/=3GPu. While yeast U1 snRNA has three matches to the Sm consensus, the U1 3'-terminal Sm site was found to be both necessary and sufficient for U1 function. Mutation of this site inhibited pre-mRNA splicing, blocked cell division and resulted in the accumulation of two 3'-extended forms of the U1 snRNA. Cells which harbor the Sm site mutation lack mature U1 RNA (U1alpha) but have a minor polyadenylated species, U1gamma, and a prominent, non-polyadenylated species, U1beta. Metabolic depletion of the essential Sm core protein, Smd1p, also resulted in the increased accumulation of U1beta and U1gamma. In vitro, synthetic U1 precursors were cleaved by Rnt1p (RNase III) very near the U1beta 3'-end observed in vivo. We propose that U1beta is an Rnt1p-cleaved intermediate and that U1 maturation to the U1alpha form occurs through an Sm-sensitive step. Interestingly, both U1alpha and a second, much longer RNA, U1straightepsilon, were produced in an rnt1 mutant strain. These results suggest that yeast U1 snRNA processing may progress through Rnt1p-dependent and Rnt1p-independent pathways, both of which require a fun-ctional Sm site for final snRNA maturation.

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.

Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins.

The U1 snRNP functions to nucleate spliceosome assembly on newly transcribed pre-mRNA. Saccharomyces cerevisiae is unusual among eukaryotes in the greatly extended length of its U1 snRNA and the apparent increased polypeptide complexity of the corresponding U1 snRNP. In this paper, we report the identification of a novel U1 snRNP protein, Prp42p, with unexpected properties. Prp42p was identified by its surprising structural similarity to the essential U1 snRNP protein, Prp39p. Both Prp39p and Prp42p possess multiple copies of a variant tetratricopeptide repeat, an element implicated in a wide range of protein assembly events. Yeast strains depleted of Prp42p by transcriptional repression of a GAL1::PRP42 fusion gene arrest for splicing prior to pre-mRNA 5' splice site cleavage. Prp42p was not observed in a recent biochemical analysis of purified U1 snRNPs from S. cerevisiae (28). Nevertheless, antibodies directed against an epitope-tagged version of Prp42p specifically precipitate U1 snRNA from yeast extracts. Furthermore, Prp42p is required for U1 snRNP biogenesis, because yeast strains depleted of Prp42p formed incomplete U1 snRNPs that failed to produce stable complexes with pre-mRNA in vitro. The evidence shows that Prp39p and Prp42p are both required to configure the atypical yeast U1 snRNP into a structure compatible with its evolutionarily conserved role in pre-mRNA splicing.

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

Prevalence and distribution of introns in non-ribosomal protein genes of yeast.

Relatively few genes in the yeast Saccharomyces cerevisiae are known to contain intervening sequences. As a group, yeast ribosomal protein genes exhibit a higher prevalence of introns when compared to non-ribosomal protein genes. In an effort to quantify this bias we have estimated the prevalence of intron sequences among non-ribosomal protein genes by assessing the number of prp2-sensitive mRNAs in an in vitro translation assay. These results, combined with an updated survey of the GenBank DNA database, support an estimate of 2.5% for intron-containing non-ribosomal protein genes. Furthermore, our observations reveal an intriguing distinction between the distributions of ribosomal protein and non-ribosomal protein intron lengths, suggestive of distinct, gene class-specific evolutionary pressures.

Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.

The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related observations, we propose that Prp39p functions, at least in part, prior to stable branch point recognition by the U1 snRNP particle to facilitate or stabilize the U1 snRNP/5' splice site interaction.

Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences.

Parallel investigations of yeast and metazoan pre-mRNA splicing have documented enormous complexity in the nucleic acid and protein components of the cellular splicing apparatus, the spliceosome. The degree to which yeast and metazoan spliceosomal proteins differ in composition and structure is currently unknown. In this report we demonstrate that the human small nuclear ribonucleoprotein (snRNP) polypeptide D1 complements the cell lethality, splicing deficiency, and snRNA instability phenotypes associated with a yeast smd1 null allele. Mutational analysis of yeast SMD1, guided by a comparison of the predicted yeast and human proteins, reveals that a large, nonconserved portion of Smd1p is dispensable for biological activity. These observations firmly establish D1 as an essential component of the cellular splicing apparatus and suggest that yeast and metazoa are remarkably similar in the polypeptides guiding early snRNP assembly.

Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles.

The PRP38 gene of Saccharomyces cerevisiae is necessary for the excision of intron sequences from pre-mRNA and required for the maintenance of maximal levels of U6 small nuclear RNA (snRNA). This report describes the identification of a gene of related function, SMD1, located immediately 3' to PRP38. The PRP38 and SMD1 transcription units are configured in an unusual "tail-to-tail" arrangement with their respective open reading frames terminating on opposite strands of a common 6-bp region. The predicted SMD1 polypeptide, Smd1p, is 40% identical to the D1 protein of human small nuclear ribonucleoprotein particles. Experimentally induced depletion of Smd1p blocks the first step of splicing and results in growth arrest. In addition, the levels of the trimethylguanosine-capped spliceosomal snRNAs, U1, U2, U4, and U5, but not the Prp38p-sensitive U6 snRNA, decrease in response to Smd1p depletion. The cap structures of snRNAs persisting in the absence of SMD1 expression appear to be peculiar, as they are poorly recognized by an anti-trimethylguanosine antibody. These data establish Smd1p as a required component of the cellular splicing apparatus and a factor in snRNA maturation and stability.

PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.

U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection.

To address the mechanisms that underlie splice site selection and splice site partner assignment, we analyzed the splicing of yeast (Saccharomyces cerevisiae) transcripts containing splice site region duplications. When the 5'-splice site region was duplicated, both sites were utilized to the same extent, indicating little or no influence of proximity on 5'-splice site choice. However, the effect of a 5'-mutant site was greatly enhanced by the presence of an adjacent wild-type site, and this effect was reversed by the restoration of base-pairing with U1 snRNA. 3'-Splice site choice was apparently influenced by proximity, as the site closest to the 5'-splice site was greatly preferred. Studies with strains carrying some U1 snRNA mutations showed an increase in the use of the distal 3'-splice site, indicating a role for U1 snRNP in 3'-splice site selection. The data are compared with those from mammalian splice site choice experiments and suggest mechanisms that influence differential splice site choice as well as exon skipping.

Identification of sites of pre-MRNA/spliceosome association.

RNase H and synthetic DNA oligonucleotides were used to analyze the ribonucleoprotein (RNP) structure of the yeast spliceosome and to assay the pre-mRNA sequence requirements for step 1 of splicing. The data suggest that tight, stable contacts between the pre-mRNA and the spliceosome may be limited to the 5' splice site and branch point regions of the intron. A 30 nucleotide segment 3' of the branch point was found to be necessary for spliceosome maturation and essential for step 1 of splicing. Somewhat surprisingly, the 3' splice site was sensitive to nuclease digestion and completely dispensable for step 1 of splicing.

A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing.

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.

S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA.

We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.

A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae.

To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box. These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract. The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events. Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al. 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect. The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs. The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.

Differential nuclease sensitivity identifies tight contacts between yeast pre-mRNA and spliceosomes.

The oligonucleotide-directed RNase H sensitivity of a yeast (Saccharomyces cerevisiae) pre-mRNA was determined in an in vitro splicing reaction. While most of the pre-mRNA was sensitive to cleavage, the regions of the 5' splice site and TACTAAC box were found to be highly resistant. The biochemical requirements for protection against nuclease attack parallel those of both spliceosome formation and splicing. Most of the uncleaved pre-mRNA remaining after RNase H challenge was found associated with two forms of the yeast spliceosome. Differences in the RNase H sensitivity of pre-mRNA found in the two spliceosome forms indicate an increased association of splicing factors with the 5' splice site during spliceosome assembly.

Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes.

Three splicing complexes formed with a yeast pre-messenger RNA during in vitro splicing can be resolved by non-denaturing gel electrophoresis after incubation in the presence of non-specific competitor RNA. The time course of the appearance of these complexes and their composition suggest that they represent an ordered pathway of splicing complex assembly.

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing.

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.