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.     

The human U1-70K snRNP protein: cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding.

We have isolated and sequenced cDNA clones encoding the human U1-70K snRNP protein, and have mapped this locus (U1AP1) to human chromosome 19. The gene produces two size classes of RNA, a major 1.7-kb RNA and a minor 3.9-kb RNA. The 1.7-kb species appears to be the functional mRNA; the role of the 3.9-kb RNA, which extends further in the 5' direction, is unclear. The actual size of the hU1-70K protein is probably 52 kd, rather than 70 kd. The protein contains three regions similar to known nucleic acid-binding proteins, and it binds RNA in an in vitro assay. Comparison of the cDNA sequences indicates that there are multiple subclasses of mRNA that arise by alternative pre-mRNA splicing of at least four alternative exon segments. This suggests that multiple forms of the hU1-70K protein may exist, possibly with different functions in vivo.     

A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression

The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and 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.     

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.     

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5′ splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.     

Xylem sap protein composition is conserved among different plant species

Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 g ml^–1. One-dimensional gel electrophoresis (SDS–PAGE) resulted in 10–20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.Abbreviations CHT Chitinase - 1D One-dimensional - GRP Glycine-rich protein - SP Serine protease - SSP Subtilisin-like serine protease - POX Peroxidase     

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 role of U5 snRNP in pre-mRNA splicing.

The current model for the function of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome proposes that U5 carries binding sites for the 5' and 3' exons, allowing the spliceosome to 'tether' the 5' exon intermediate produced by the first catalytic step and align it with the 3' exon for the second step. Functional analysis of U5 snRNA in cis-spliceosomes has provided support for this model, and data from nematode and trypanosome splicing systems suggest that U5 or a U5-like snRNA performs a similar role in trans-splicing.     

Cloning of a yeast U1 snRNP 70K protein homologue: functional conservation of an RNA-binding domain between humans and yeast.

We have cloned and sequenced a gene encoding a yeast homologue of the U1 snRNP 70K protein. The gene, SNP1, encodes a protein which has 30% amino acid identity with the human 70K protein and has a predicted molecular weight of 34 kDa. The yeast and human sequences are more closely related to each other than to other (non-U1) RNA-binding proteins, but diverge considerably in their C-terminal portions. In particular, SNP1 lacks the charged carboxy terminus of the human 70K protein. A yeast strain, a alpha 115, was constructed in which one allele of the SNP1 gene contained a 554 bp deletion. Tetrad analysis of a alpha 115 showed that the SNP1 gene is essential for the viability of yeast cells. The complete human 70K gene did not complement snp1, but the lethal snp1 mutation was rescued by plasmids bearing a chimera in which over half the yeast gene was replaced with the homologous region of the human 70K gene, including the RNA-binding domain. These results suggest that SNP1 encodes a functional homologue of the U1 snRNP 70K protein.     

Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators.

SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner. This latter property suggests a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing. Human SF2 cDNAs have been isolated and overexpressed in bacteria. Recombinant SF2 is active in splicing and stimulates proximal 5' splice sites. SF2 has a C-terminal region rich in arginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70K polypeptide and the Drosophila alternative splicing regulators transformer, transformer-2, and suppressor-of-white-apricot. Like transformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.     

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins (su(wa) and tra) that may regulate alternative pre-messenger RNA splicing.     

Protein transport into mitochondria is conserved between plant and yeast species

Protein targeting into plant mitochondria was investigated by in vitro translocation experiments. The precursor of the mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia was synthesized in vitro, translocated to, processed, and assembled in purified Vicia faba mitochondria. Transport (but not binding) required a membrane potential and external nucleotides and was conserved among plant species. beta subunit precursors from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe were imported and correctly processed in plant mitochondria. This translocation used protease-sensitive components of the outer membrane. Conversely, the N. plumbaginifolia beta subunit precursor was efficiently translocated and cleaved in yeast mitochondria. However, a precursor for a chloroplast protein was not targeted to plant or yeast mitochondria. We conclude that the machinery for protein import into mitochondria is specific and conserved in plant and yeast organisms. These results are discussed in the context of a poly- or monophyletic origin of mitochondria.     

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.     

Isoforms of U1-70k Control Subunit Dynamics in the Human Spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B''. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B'' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.