The amino-terminal domain of yeast U1-70K is necessary and sufficient for function.

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.     

The yeast homolog of the U1 snRNP protein 70K is encoded by the SNP1 gene.

The product of the yeast SNP1 gene has high homology to two domains of the metazoan U1 snRNP protein 70K, which binds to stem/loop I of the U1 RNA. However, the absence of other domains conserved in metazoan 70K and the minimal effect of yeast U1 RNA stem/loop I deletion make the assignment of SNP1 as yeast 70K less clear. To address this question, we have expressed the SNP1 gene as a fusion protein in E. coli and developed a gel shift assay for U1 RNA binding. We show here that the product of the yeast SNP1 gene binds directly and specifically to the first 47 nucleotides of yeast U1 RNA, which include the stem/loop 1 structure. We therefore conclude that the SNP1 gene product is the yeast 70K homolog. This is the first yeast protein to be identified as a homolog of a metazoan snRNP protein.     

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

Drosophila SNF/D25 combines the functions of the two snRNP proteins U1A and U2B' that are encoded separately in human, potato, and yeast.

The plant and vertebrate snRP proteins U1A and U2B' are structurally closely related, but bind to different U snRNAs. Two additional related snRNP proteins, the yeast U2B' protein and Drosophila SNF/D25 protein, are analyzed here. We show that the previously described yeast open reading frame YIB9w encodes yeast U2B' as judged by the fact that the protein encoded by YIB9w bindsto stem-loop IV of yeast U2 snRNA in vitro and is part of the U2 snRNP in vivo. In contrast to the human U2B' protein, specific binding of yeast U2B' to RNA in vitro can occur in the absence of an accessory U2A' protein. The Drosophila SNF-D25 protein, unlike all other U1A/U2B' proteins studied to date, is shown to be a component of both U1 and U2 snRNPs. In vitro, SNF/D25 binds to U1 snRNA on itsown and to U2 snRNA in the presence of either the human U2A' protein or of Drosophila nuclear extract. Thus, its RNA-binding properties are the sum of those exhibited by human or potato U1A and U2B' proteins. Implications for the role of SNF/D25 in alternative splicing, and for the evolution of the U1A/U2B' protein family, are discussed.     

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.     

Human U1-70K ribonucleoprotein antigen gene: organization, nucleotide sequence, and mapping to locus 19q13.3

We have isolated and sequenced the gene encoding the human U1-70K snRNP protein. U1-70K is an RNA-binding protein that is a specific component of the U1 small nuclear ribonucleoprotein complex (snRNP) and constitutes the major anti-(U1) RNP autoimmune antigen. We have mapped the U1-70K gene to the distal portion of chromosome 19, at band q13.3. The gene is greater than 44 kb in size and consists of 11 exons. The general structure of the gene has been completely conserved during vertebrate evolution and accounts for the production of several different U1-70K mRNA species by alternative pre-mRNA splicing. Comparison of the predicted amino acid sequences of animal U1-70K proteins reveals a high degree of conservation, particularly in the region of the RNP consensus domain. Even more striking is the complete conservation of the nucleotide sequence of an alternative included/excluded exon containing an in-frame translational termination codon. This conservation also includes significant portions of the downstream intervening sequence. This extraordinary conservation at the nucleotide sequence level suggests that alternative splicing of this exon serves an important function, perhaps in regulating the production of functional U1-70K protein.     

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.     

Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA.

We have studied the interaction of two of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins, U1-70K and U1-A, with U1 small nuclear RNA (snRNA). The U1-70K protein is a U1-specific RNA-binding protein. Deletion and mutation analyses of a beta-galactosidase/U1-70K partial fusion protein indicated that the central portion of the protein, including the RNP sequence domain, is both necessary and sufficient for specific U1 snRNA binding in vitro. The highly conserved eight-amino-acid RNP consensus sequence was found to be essential for binding. Deletion and mutation analyses of U1 snRNA showed that both the U1-70K fusion protein and the native HeLa U1-70K protein bound directly to loop I of U1 snRNA. Binding was sequence specific, requiring 8 of the 10 bases in the loop. The U1-A snRNP protein also interacted specifically with U1 snRNA, principally with stem-loop II.     

The Drosophila U1-70K protein is required for viability, but its arginine-rich domain is dispensable

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain's importance in intact multicellular organisms have not been carried out. Here we report a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, we find that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, we show that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together our studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function.     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.     

Characterization of yeast U1 snRNP A protein: identification of the N-terminal RNA binding domain (RBD) binding site and evidence that the C-terminal RBD functions in splicing.

The yeast U1A protein is a U1 snRNP-specific protein. Like its human counterpart (hU1A), it has two conserved RNA binding domains (RBDs). The N-terminal RBD is quite different from the human protein, and a binding site on yeast U1 snRNA is not readily apparent. The C-terminal RBD is of unknown function. Using in vivo dimethyl sulfate (DMS) protection of mutant strains, we defined a region in yeast U1 snRNA as the likely U1A N-terminal RBD binding site. This was confirmed by direct in vitro binding assays. The site is very different from its vertebrate counterpart, but its location within yeast U1 snRNA suggests a conserved structural relationship to other U1 snRNP components. Genetic studies and sensitive in vivo splicing measurements indicate that the yeast U1A C-terminal RBD also functions in pre-mRNA splicing. We propose that the N-terminal RBD serves to tether the splicing-relevant C-terminal RBD to the snRNP.     

The yeast U2A''/U2B complex is required for pre-spliceosome formation.

Human U2 snRNP contains two specific proteins, U2A' and U2B", that interact with U2 snRNA stem-loop IV. In Saccharomyces cerevisiae, only the counterpart of human U2B", Yib9p, has been identified. Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A', henceforth called LEA1 (looks exceptionally like U2A'). We demonstrate that Lea1p is a specific component of the yeast U2 snRNP. In addition, we show that Lea1p interacts directly with Yib9p. In vivo association of Lea1p with U2 snRNA requires Yib9p. Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem-loop IV in vitro. Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA. Pre-mRNA splicing is strongly impaired in these cells. In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP. This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s). This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome.     

The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression.

The dbf3 mutation was originally obtained in a screen for DNA synthesis mutants with a cell cycle phenotype in the budding yeast Saccharomyces cerevisiae. We have now isolated the DBF3 gene and found it to be an essential gene with an ORF of 7239 nucleotides, potentially encoding a large protein of 268 kDa. We also obtained an allele-specific high copy number suppressor of the dbf3-1 allele, encoded by the known SSB1 gene, a member of the Hsp70 family of heat shock proteins. The sequence of the Dbf3 protein is 58% identical over 2300 amino acid residues to a predicted protein from Caenorhabditis elegans. Furthermore, partial sequences with 61% amino acid sequence identity were deduced from two files of human cDNA in the EST nucleotide database so that Dbf3 is a highly conserved protein. The nucleotide sequence of DBF3 turned out to be identical to the yeast gene PRP8, which encodes a U5 snRNP required for pre-mRNA splicing. This surprising result led us to further characterise the phenotype of dbf3 which confirmed its role in the cell cycle and showed it to function early, around the time of S phase. This data suggests a hitherto unexpected link between pre-mRNA splicing and the cell cycle.     

The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.     

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.