A splicing factor, Prp8: preferential localization in the testis and ovary in adult mice

Prp8 is a splicing factor of 220 kDa originally identified in yeast and is a component of the U5 small nuclear ribonucleoprotein particle. Mouse Prp8 cDNA was cloned and shown to share 62.6 and 68.2% sequence identity with the yeast homologue at the amino acid and nucleotide level, respectively, while it differs by only 3 amino acid residues from the human homologue. During mouse embryogenesis, Prp8 is expressed intensely at day 9.5 of gestation, and its expression decreases progressively during embryogenesis. In adult mice, Prp8 is expressed strongly in the testis and moderately in the ovary. in situ hybridization analysis revealed that Prp8 is preferentially expressed in the outer cell layer in the testis, probably in the spermatogonia and primary spermatocytes, and in granulosa cells in the ovary. In Caenorhabditis elegans, microinjection of a double stranded RNA corresponding to a portion of the Prp8 sequence results in the arrest of embryogenesis at the late-gastrulation stage. These results suggest that Prp8 plays an important role in reproduction and development. Prp8 was shown to bind to midkine (MK), a heparin-binding growth factor. Since Prp8 expression partially overlaps with the sites of action of MK, it is possible that binding to Prp8 is involved in part of MK signaling.     

The Prp1(+) Gene Required for Pre-mRNA Splicing in Schizosaccharomyces Pombe Encodes a Protein That Contains Tpr Motifs and Is Similar to Prp6p of Budding Yeast

The prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe have a defect in pre-mRNA splicing and accumulate mRNA precursors at a restrictive temperature. One of the prp mutants, prp1-4, also has a defect in poly(A)(+) RNA transport. The prp1(+) gene encodes a protein of 906 amino acid residues that contains 19 repeats of 34 amino acids termed tetratrico peptide repeat (TPR) motifs, which were proposed to mediate protein-protein interactions. The amino acid sequence of Prp1p shares 29.6% identity and 50.6% similarity with that of the PRP6 protein of Saccharomyces cerevisiae, which is a component of the U4/U6 snRNP required for spliceosome assembly. No functional complementation was observed between S. pombe prp1(+) and S. cerevisiae PRP6. We examined synthetic lethality of prp1-4 with the other known prp mutations in S. pombe. The results suggest that Prp1p interacts either physically or functionally with Prp4p, Prp6p and Prp13p. Interestingly, the prp1(+) gene was found to be identical with the zer1(+) gene that functions in cell cycle control. These results suggest that Prp1p/Zer1p is either directly or indirectly involved in cell cycle progression and/or poly(A)(+) RNA nuclear export, in addition to pre-mRNA splicing.     

Cef1p is a component of the Prp19p-associated complex and essential for pre-mRNA splicing

The Prp19p protein of the budding yeast Saccharomyces cerevisiae is an essential splicing factor and is associated with the spliceosome during the splicing reaction. We have previously shown that Prp19p is not tightly associated with small nuclear ribonucleoprotein particles but is associated with a protein complex consisting of at least eight protein components. By sequencing components of the affinity-purified complex, we have identified Cef1p as a component of the Prp19p-associated complex, Ntc85p. Cef1p could directly interact with Prp19p and was required for pre-mRNA splicing both in vivo and in vitro. The c-Myb DNA binding motif at the amino terminus of Cef1p was required for cellular growth but not for interaction of Cef1p with Prp19p or Cef1p self-interaction. We have identified a small region of 30 amino acid residues near the carboxyl terminus required for both cell viability and protein-protein interactions. Cef1p was associated with the spliceosome in the same manner as Prp19p, i.e. concomitant with or immediately after dissociation of U4. The anti-Cef1p antibody inhibited binding to the spliceosome of Cef1p, Prp19p, and at least three other components of the Prp19p-associated complex, suggesting that the Prp19p-associated complex is likely associated with the spliceosome and functions as an integral complex.     

Drosophila MFAP1 is required for pre-mRNA processing and G2/M progression

The mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions ( Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14 ). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component ( Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938-2946 ), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G(2)/M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G(2)/M arrest might be a general consequence of interfering with spliceosome function.     

Crystal structure of the human U4/U6 small nuclear ribonucleoprotein particle-specific SnuCyp-20, a nuclear cyclophilin

The cyclophilin SnuCyp-20 is a specific component of the human U4/U6 small nuclear ribonucleoprotein particle involved in the nuclear splicing of pre-mRNA. It stably associates with the U4/U6-60kD and -90kD proteins, the human orthologues of the Saccharomyces cerevisiae Prp4 and Prp3 splicing factors. We have determined the crystal structure of SnuCyp-20 at 2.0-A resolution by molecular replacement. The structure of SnuCyp-20 closely resembles that of human cyclophilin A (hCypA). In particular, the catalytic centers of SnuCyp-20 and hCypA superimpose perfectly, which is reflected by the observed peptidyl-prolyl-cis/trans-isomerase activity of SnuCyp-20. The surface properties of both proteins, however, differ significantly. Apart from seven additional amino-terminal residues, the insertion of five amino acids in the loop alpha1-beta3 and of one amino acid in the loop alpha2-beta8 changes the conformations of both loops. The enlarged loop alpha1-beta3 is involved in the formation of a wide cleft with predominantly hydrophobic character. We propose that this enlarged loop is required for the interaction with the U4/U6-60kD protein.     

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex

PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). We have then shown that PAP-1 plays a role in pre-mRNA splicing. Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study. The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding. Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome. These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.     

Genetic and functional interaction of evolutionarily conserved regions of the Prp18 protein and the U5 snRNA

Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.     

A new cyclophilin and the human homologues of yeast Prp3 and Prp4 form a complex associated with U4/U6 snRNPs.

We have purified three new human U4/U6-snRNP proteins from HeLa cells. The three proteins formed a tightly bound complex and behaved as a single species throughout the purification. All three proteins have been identified by peptide sequencing, and full-length cDNA sequences have been obtained for all of them. Two of the proteins are homologues of the Saccharomyces cerevisiae splicing factors Prp3 and Prp4, and the third protein is a cyclophilin. Both the human and S. cerevisiae Prp4 proteins have seven repeats of the WD motif and likely fold into structures very similar to those of the beta subunits of G proteins. The human Prp3 protein is highly basic and is closely related to S. cerevisiae Prp3 only in its carboxyl-terminal half. The human homologues of Prp3 and Prp4 are part of a stable complex in the absence of RNA. The third protein in the complex is a new cyclophilin. Cyclophilins have been proposed to act as chaperones in a variety of cellular processes, and we discuss some possible roles of this U4/U6 snRNP-associated cyclophilin.     

Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements.

U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.     

Structure and assembly of the SF3a splicing factor complex of U2 snRNP

SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structure-guided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.     

Functional and physical interaction between the yeast splicing factors Slu7 and Prp18.

We show that the requirement for Prp18 during the second step of actin pre-mRNA splicing in vitro is dictated by the distance between the branch point and the 3'splice site. Prp18 is dispensable for splicing of precursor RNAs in which the interval between the branch point and 3'splice site is <12 nt. This resembles the requirement for another second step factor, Slu7. Excess Slu7 protein can bypass the need for Prp18 in vitro , suggesting that Slu7 and Prp18 function in a concerted manner. Physical interaction between Slu7 and Prp18 was demonstrated by using the two-hybrid assay. Deletion mutants of SLU7 were tested for their ability to support growth of a slu7 null strain. Removal of 199 amino acids from the N-terminus of the 382 amino acid Slu7 protein did not affect cell viability at 25 degrees C. A more extensive N-terminal deletion of 221 amino acids was lethal, as was a C-terminal deletion of 47 amino acids. Deleted versions of Slu7 were also tested for interaction with Prp18 in the two-hybrid system. We define a segment of Slu7 from residue 200 to 224 that is necessary for interaction with Prp18.     

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.     

Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.     

Definition of a spliceosome interaction domain in yeast Prp2 ATPase

The Saccharomyces cerevisiae splicing factor Prp2 is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.     

A novel mammalian nuclear protein similar to Schizosaccharomyces pombe Prp1p/Zer1p and Saccharomyces cerevisiae Prp6p pre-mRNA splicing factors

We have cloned a novel 100-kDa mammalian protein, which was recognized by an anti-peptide antibody against an epitope-containing nuclear localization signal of NF-kappaB p65 subunit. Predicted amino acid sequence of the protein is similar to those of yeast splicing factors, Prp1p/Zer1p of Schizosaccharomyces pombe and Prp6p of Saccharomyces cerevisiae. Among these proteins, tetratrico peptide repeat (TPR) motif, which mediates protein-protein interactions, is conserved, whereas leucine zipper motif is found only in the 100-kDa protein. Indirect immunofluorescent staining showed that the 100-kDa protein localized in the nucleus in HeLa cells.     

Identification and characterisation of Schizosaccharomyces pombe cyclophilin 3, a cyclosporin A insensitive orthologue of human USA-CyP

We have identified nine cyclophilins encoded in the genome of the fission yeast Schizosaccharomyces pombe (Sp). Cyclophilin 3 is an orthologue of hUSA-CyP, which is associated with Prp4/Prp3 in the [U4/U6.U5] snRNP complex and Prp18, both of which are components of the pre-mRNA splicing machinery. PPIase assays have shown SpCyp3 and hUSA-CyP to have comparable activity and substrate specificity, but SpCyp3 has a reduced sensitivity to CsA correlating with a difference in the catalytic site. Prp3, Prp4 and Prp18 proteins exist in S. pombe and nuclear localisation of SpCyp3 has been shown, indicating conservation of function between hUSA-CyP and SpCyp3.     

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.     

Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p

The U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) are components of the spliceosome, which catalyzes pre-mRNA splicing. One of the largest and the most highly conserved proteins in the spliceosome is Prp8p, a component of the U5 snRNP. Despite its size and conservation, very few motifs have been identified that suggest specific biochemical functions. A variant of the Jab1/MPN domain found in a class of deubiquitinating enzymes is present near the C terminus of Prp8p. Ubiquitination regulates a broad range of cellular pathways, and its functions generally require ubiquitin recognition by one or more ubiquitin-binding domains (UBDs). No precise role for ubiquitin has been defined in the pre-mRNA splicing pathway, and no known UBDs have been found within splicing proteins. Here we show that a Prp8p fragment containing the Jab1/MPN domain binds directly to ubiquitin with an affinity comparable to other known UBDs. Several mutations within this domain that compromise splicing also reduce interaction of the fragment with ubiquitin-Sepharose. Our results define a new UBD and suggest functional links between ubiquitin and the pre-mRNA splicing machinery.