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.     

Fas (APO-1/CD95)-mediated apoptosis is independent of bcl-2: a study with cell lines overexpressing bcl-2 and with bcl-2 transfected cell lines

Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.     

Modulation of E-cadherin expression in TPA-induced cell motility: well-differentiated human adenocarcinoma cells move as coherent sheets associated with phosphorylation of E-cadherin-catenin complex

We have identified a human splicing factor required for the second step of pre-mRNA splicing. This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18. In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished. Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step. The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing. hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans. Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved. A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes. The homology identifies one region important for the function of both proteins and may define a new protein motif. In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs. A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.     

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.     

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.     

U1 small nuclear ribonucleoprotein particle-protein interactions are revealed in Saccharomyces cerevisiae by in vivo competition assays

Two highly conserved regions of the 586-nucleotide yeast (Saccharomyces cerevisiae) U1 small nuclear RNA (snRNA) can be mutated or deleted with little or no effect on growth rate: the universally conserved loop II (corresponding to the metazoan A loop) and the yeast core region (X. Liao, L. Kretzner, B. Séraphin, and M. Rosbash, Genes Dev. 4:1766-1774, 1990). To examine the contribution of these regions to U1 small nuclear ribonucleoprotein particle (snRNP) activity, a competitor U1 gene, encoding a nonfunctional U1 snRNA molecule, was introduced into a number of strains carrying a U1 snRNA gene with loop II or yeast core mutations. The presence of the nonfunctional U1 gene lowered the growth rate of these mutant strains but not wild-type strains, consistent with the notion that mutant U1 RNAs are less active than wild-type U1 snRNAs. A detailed analysis of the U1 snRNA levels and half-lives in a number of merodiploid strains suggests that these mutant U1 snRNAs interact with U1 snRNP proteins less well than do their wild-type counterparts. Competition for protein factors during snRNP assembly could account for a number of previous observations in both yeast and mammalian cells.     

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.     

A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import. Previous studies have demonstrated that the assembly of pre-snRNAs into ribonucleoprotein particles containing the Sm core proteins is essential for nuclear import in mammalian cells but that 5' cap hypermethylation is not. In the present investigation we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' tails, and identified one that was stalled (or greatly slowed) in 3' end processing, leading to its accumulation in the cytoplasm of human cells. Nonetheless, this 3' processing arrested pre-U2 RNA molecule was found to undergo cytoplasmic assembly into Sm protein-containing complexes to the same extent as normal pre-U2 RNA. The Sm protein-associated, unprocessed mutant pre-U2 RNA was not observed in the nuclear fraction. Using an assay based on suppression of a genetically blocked SV40 pre-mRNA splicing pathway, we found that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.     

An unusual chemical reactivity of Sm site adenosines strongly correlates with proper assembly of core U snRNP particles

The small nuclear ribonucleoprotein particles (snRNP) U1, U2, U4, and U5 contain a common set of eight Sm proteins that bind to the conserved single-stranded 5'-PuAU3-6GPu-3' (Sm binding site) region of their constituent U snRNA (small nuclear RNA), forming the Sm core RNP. Using native and in vitro reconstituted U1 snRNPs, accessibility of the RNA within the Sm core RNP to chemical structure probes was analyzed. Hydroxyl radical footprinting of in vitro reconstituted U1 snRNP demonstrated that riboses within a large continuous RNA region, including the Sm binding site, were protected. This protection was dependent on the binding of the Sm proteins. In contrast with the riboses, the phosphate groups within the Sm core site were accessible to modifying reagents. The invariant adenosine residue at the 5' end, as well as an adenosine two nucleotides downstream of the Sm binding site, showed an unexpected reactivity with dimethyl sulfate. This novel reactivity could be attributed to N7-methylation of the adenosine and was not observed in naked RNA, indicating that it is an intrinsic property of the RNA- protein interactions within the Sm core RNP. Further, this reactivity was observed concomitantly with formation of the Sm subcore intermediate during Sm core RNP assembly. As the Sm subcore can be viewed as the commitment complex in this assembly pathway, these results suggest that the peculiar reactivity of the Sm site adenosine bases may be diagnostic for proper assembly of the Sm core RNP. Consistent with this idea, a strong correlation was found between the unusual N7-A methylation sensitivity of the Sm core RNP and its ability to be imported into the nucleus of Xenopus laevis oocytes.     

A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles

The spliceosome removes introns from pre-messenger RNAs by a mechanism that entails extensive remodeling of RNA structure. The most conspicuous rearrangement involves disruption of 24 base pairs between U4 and U6 small nuclear RNAs (snRNAs). Here, the yeast RNA binding protein Prp24 is shown to reanneal these snRNAs. When Prp24 is absent, unpaired U4 and U6 small nuclear ribonucleoprotein particles (snRNPs) accumulate; with time, splicing becomes inhibited. Addition of purified Prp24 protein regenerates duplex U4/U6 snRNPs for new rounds of splicing. The reannealing reaction catalyzed by Prp24 proceeds more efficiently with snRNPs than with deproteinized snRNAs.     

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.     

The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins

Spinal muscular atrophy (SMA), one of the most common fatal autosomal recessive diseases, is characterized by degeneration of motor neurons and muscular atrophy. The SMA disease gene, termed Survival of Motor Neurons (SMN), is deleted or mutated in over 98% of SMA patients. The function of the SMN protein is unknown. We found that SMN is tightly associated with a novel protein, SIP1, and together they form a specific complex with several spliceosomal snRNP proteins. SMN interacts directly with several of the snRNP Sm core proteins, including B, D1-3, and E. Interestingly, SIP1 has significant sequence similarity with Brr1, a yeast protein critical for snRNP biogenesis. These findings suggest a role for SMN and SIP1 in spliceosomal snRNP biogenesis and function and provide a likely molecular mechanism for the cause of SMA.     

Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor

Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing. In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3. We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions. However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal half to the beta-subunits of G proteins. We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed beta-propeller based on the crystalline structure of beta-transducin. Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the beta-strands of the propeller. This surface could be used as a scaffold for generating an RNA-protein complex.     

Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron

Nuclear pre-messenger RNA splicing requires the action of five small nuclear (sn) RNAs, U1, U2, U4, U5 and U6, and more than 50 proteins. The mechanistic similarity of nuclear pre-mRNA splicing and group II self-splicing suggests that many of the central processes of nuclear pre-mRNA splicing are based on RNA-RNA interaction. To understand the mechanism of pre-mRNA splicing, the interactions, and their temporal relationships, that occur between the snRNAs and the pre-mRNA during splicing must be identified. Several snRNA-snRNA and snRNA-intron interactions have been demonstrated but the putative RNA-based interactions that recognize the AG dinucleotide at the 3' splice site during 3' cleavage and exon ligation are unknown. We report here the reciprocal suppression between 5' and 3' splice site mutations in the yeast actin intron, and propose that the 3' splice site is positioned for 3' cleavage and exon ligation, at least in part, through a non-Watson-Crick interaction between the guanosines at the 5' and 3' splice sites.     

The human U5-220kD protein (hPrp8) forms a stable RNA-free complex with several U5-specific proteins, including an RNA unwindase, a homologue of ribosomal elongation factor EF-2, and a novel WD-40 protein

The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, protein-protein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.     

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.     

A coactivator of pre-mRNA splicing

The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.     

The pre-mRNA 5' cap determines whether U6 small nuclear RNA succeeds U1 small nuclear ribonucleoprotein particle at 5' splice sites

Efficient splicing of the 5'-most intron of pre-mRNA requires a 5' m7G(5')ppp(5')N cap, which has been implicated in U1 snRNP binding to 5' splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5' cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5' splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5' splice site and not with any loss of U1 snRNP binding.     

Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis

The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.