U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle.

U4 and U6 RNAs of mammalian cells possess extensive intermolecular sequence complementarity and hence have the potential to base pair. A U4/U6 RNA complex, detectable in nondenaturing polyacrylamide gels, is released when human small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 RNAs are dissociated with proteinase K in the presence of sodium dodecyl sulfate. The released RNA/RNA complex dissociates with increasing temperature, consistent with the existence of specific base-pairing between the two RNAs. Since U6 RNA is selectively released from intact snRNPs under the same conditions required to dissociate the U4/U6 RNA complex, the RNA-RNA interaction may be sufficient to maintain U4 and U6 RNAs in the same snRNP particle. The biological implications of these findings are discussed.     

Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions.

Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.     

Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein

Selective cleavage of U4 or U6 RNA in a HeLa cell nuclear extract inhibits splicing of pre-mRNAs containing an adenovirus or a simian virus 40 intron. RNAs in the U4/U6 small nuclear ribonucleoprotein (snRNP) were specifically degraded with RNAase H and deoxyoligonucleotides. Two oligomers complementary to U4 RNA and two complementary to U6 RNA cleave their target RNAs and inhibit the appearance of both spliced products and reaction intermediates. Splicing is reconstituted by mixing an extract containing cleaved U4 or U6 RNA with one in which splicing has been inhibited by degrading U2 RNA. All four abundant snRNPs, containing U1, U2, U5, or U4 and U6 RNAs, are now implicated in pre-mRNA splicing. Possible interactions of the U4/U6 snRNP with other components of the splicing complex are discussed.     

The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent spliceosome

U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5' splice site and branchpoint sequence. Thus, within the U12-type prespliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein-protein and protein-RNA interactions contributing to 5' splice site recognition and/or intron bridging appear to differ significantly in the minor versus major prespliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type spliceosome provides insight into potential evolutionary relationships between the major and minor spliceosome.     

Assembly and nuclear transport of the U4 and U4/U6 snRNPs

We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.     

Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides.

Immunoprecipitation of human small nuclear ribonucleoproteins (snRNPs) containing the small nuclear RNAs U1, U2, U4, U5, and U6 with two antibodies produced in certain patients suffering from systemic lupus erythematosus was used to identify the polypeptides present on human U1 and U2 snRNPs. U1 and U2 snRNPs contain both common and unique polypeptides; visualization of the differences was possible through the use of non-methionine protein labeling and partial fractionation of snRNP populations. To facilitate comparisons with results from other laboratories, we have designated the snRNP polypeptides by their molecular weights. Four small polypeptides, P8, P9, P10, and P12, of 8,000 to 12,000 daltons, are each present in equal amounts on both U1 and U2 snRNPs. U1 snRNPs also contain a unique 30,000-dalton polypeptide, P30, whereas U2 snRNPs contain a unique 27,000-dalton, methionine-deficient polypeptide, P27. A closely migrating pair of polypeptides, P23 and P22, of 23,000 and 21,500 daltons, respectively, is present on both snRNPs; U2 snRNPs are enriched in the former, and U1 snRNPs are enriched in the latter.     

U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation

The requirement for individual U RNAs in splicing and polyadenylation was investigated using oligonucleotide-directed cleavage of snRNAs in in vitro processing extracts. Cleavage of U1, U2, or U4 RNA inhibited splicing but not polyadenylation of short precursor RNAs. Thus each snRNA and the snRNP in which it is assembled participates in the splicing reaction. Splicing activity was recovered when extracts containing cleaved U RNAs were mixed in pairwise combinations, indicating that U1, U2, and U4/U6 snRNPs independently interact with the assembling spliceosome. The involvement of multiple snRNPs in the splicing of simple precursor RNAs suggests that the spliceosome is a large complex assembly consisting of multiple snRNPs whose activity is dependent on the structural integrity of the individual U RNAs.     

Recycling of the U12-type spliceosome requires p110, a component of the U6atac snRNP

U12-dependent introns are spliced by the so-called minor spliceosome, requiring the U11, U12, and U4atac/U6atac snRNPs in addition to the U5 snRNP. We have recently identified U6-p110 (SART3) as a novel human recycling factor that is related to the yeast splicing factor Prp24. U6-p110 transiently associates with the U6 and U4/U6 snRNPs during the spliceosome cycle, regenerating functional U4/U6 snRNPs from singular U4 and U6 snRNPs. Here we investigated the involvement of U6-p110 in recycling of the U4atac/U6atac snRNP. In contrast to the major U6 and U4/U6 snRNPs, p110 is primarily associated with the U6atac snRNP but is almost undetectable in the U4atac/U6atac snRNP. Since p110 does not occur in U5 snRNA-containing complexes, it appears to be transiently associated with U6atac during the cycle of the minor spliceosome. The p110 binding site was mapped to U6 nucleotides 38 to 57 and U6atac nucleotides 10 to 30, which are highly conserved between these two functionally related snRNAs. With a U12-dependent in vitro splicing system, we demonstrate that p110 is required for recycling of the U4atac/U6atac snRNP.     

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.     

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.     

In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.     

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.     

Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.     

Requirements for U2 snRNP addition to yeast pre-mRNA.

The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 snRNPs associate with the pre-mRNA prior to U5 and U4/U6 snRNPs. In yeast, U1 snRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 snRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 snRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 snRNP adds to the U1 snRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 snRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 snRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 snRNP:pre-mRNA interactions to allow subsequent U2 snRNP addition.     

U1-U2 snRNPs interaction induced by an RNA complementary to the 5'' end sequence of U1 snRNA.

Several lines of evidences indicate that U1 and U2 snRNPs become interacting during pre-mRNA splicing. Here we present data showing that an U1-U2 snRNPs interaction can be mediated by an RNA only containing the consensus 5' splice site of all of the sequences characteristic of pre-mRNAs. Using monospecific antibodies (anti-(U1) RNP and anti-(U2) RNP), we have found that a tripartite complex comprising U1 and U2 snRNPs is immunoprecipitated in the presence of a consensus 5' splice site containing RNA, either from a crude extract or from an artificial mixture enriched in U1 and U2 snRNPs. This complex does not appear in the presence of an RNA lacking the sequence complementary to the 5' terminus of U1 snRNA. Moreover, RNAse T1 protection coupled to immunoprecipitation experiments have demonstrated that only the 5' end sequence of U1 snRNA contacts the consensus 5' splice site containing RNA, arguing that U2 snRNP binding in the tripartite complex is mediated by U1 snRNP.