Evidence for a Prp24 binding site in U6 snRNA and in a putative intermediate in the annealing of U6 and U4 snRNAs.

A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes. Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.     

U4 and U6 small nuclear ribonucleoprotein particles. 2. Evidence for their involvement in pre-mRNA splicing

The in vitro splicing of pre-mRNA of the human beta-globin gene in the presence of HeLa cell nuclear extract was investigated. Splicing was inhibited by auto-antibodies against U4 and U6 snRNP particles. No intermediates or products of the splicing reaction were evident in the presence of antibodies against U4 and U6 snRNPs which suggests their involvement in pre-mRNA splicing.     

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.     

Enhancement of U4/U6 small nuclear ribonucleoprotein particle association in Cajal bodies predicted by mathematical modeling

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo specific assembly steps in Cajal bodies (CBs), nonmembrane-bound compartments within cell nuclei. An example is the U4/U6 di-snRNP, assembled from U4 and U6 monomers. These snRNPs can also assemble in the nucleoplasm when cells lack CBs. Here, we address the hypothesis that snRNP concentration in CBs facilitates assembly, by comparing the predicted rates of U4 and U6 snRNP association in nuclei with and without CBs. This was accomplished by a random walk-and-capture simulation applied to a three-dimensional model of the HeLa cell nucleus, derived from measurements of living cells. Results of the simulations indicated that snRNP capture is optimal when nuclei contain three to four CBs. Interestingly, this is the observed number of CBs in most cells. Microinjection experiments showed that U4 snRNA targeting to CBs was U6 snRNP independent and that snRNA concentration in CBs is approximately 20-fold higher than in nucleoplasm. Finally, combination of the simulation with calculated association rates predicted that the presence of CBs enhances U4 and U6 snRNP association by up to 11-fold, largely owing to this concentration difference. This provides a chemical foundation for the proposal that these and other cellular compartments promote molecular interactions, by increasing the local concentration of individual components.     

Roles of U4 and U6 snRNAs in the assembly of splicing complexes.

A series of U4 and U6 snRNA mutants was analysed in Xenopus oocytes to determine whether they block splicing complex assembly or splicing itself. All the U4 and U6 mutants found to be inactive in splicing complementation resulted in defects in assembly of either U4/U6 snRNP or of splicing complexes. No mutants were found to separate the entry of U5 and U6 snRNAs into splicing complexes and neither of these RNAs was able to associate with the pre-mRNA in the absence of U4. In the absence of U6 snRNA, however, U4 entered a complex containing pre-mRNA as well as the U1 and U2 snRNAs. U6 nucleotides whose mutation resulted in specific blockage of the second step of splicing in Saccharomyces cerevisiae are shown not to be essential for splicing in the oocyte assay. The results are discussed in terms of the roles of U4 and U6 in the assembly and catalytic steps of the splicing process.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Structure-function relationships in the vertebrate U4-U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4-U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4-U6 snRNP assembly or stability. This group includes mutations in both the proposed U4-U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4-U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.     

Characterization of the catalytic activity of U2 and U6 snRNAs

Removal of introns from pre-messenger RNAs in eukaryotes is carried out by the spliceosome, an assembly of a large number of proteins and five small nuclear RNAs (snRNAs). We showed previously that an in vitro transcribed and assembled base-paired complex of U2 and U6 snRNA segments catalyzes a reaction that resembles the first step of splicing. Upon incubation with a short RNA oligonucleotide containing the consensus sequence of the pre-mRNA branch site, the U2/U6 complex catalyzed a reaction between the 2' OH of a bulged adenosine and a phosphate in the catalytically important AGC triad of U6, leading to the formation of an X-shaped product, RNA X, apparently linked by an unusual phosphotriester bond. Here we characterize this splicing-related reaction further, showing that RNA X formation is an equilibrium reaction, and that the low yield of the reaction likely reflects an unfavorable equilibrium coefficient. Consistent with a phosphotriester linkage, RNA X is highly alkali-sensitive, but only mildly acid-sensitive. We also show that mutations in the AGC sequence of U6 can have significant effects on RNA X formation, further extending the similarities between splicing and RNA X formation. We also demonstrate that pseudouridylation of U2 enhances RNA X formation, and that U6 snRNA purified from nuclear extracts is capable of forming RNA X. Our data suggest that the ability to form RNA X might be an intrinsic property of spliceosomal snRNAs.     

PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.     

Spliceosome assembly in the absence of stable U4/U6 RNA pairing

The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6–Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6–Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6–Prp24 complex renders stable U4/U6 pairing nonessential.     

Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs.

Prp24 was previously isolated as a suppressor of a cold-sensitive U4 mutation and is required for at least the first step of splicing in vitro. Our investigation of the in vitro RNA binding properties of the purified Prp24 protein shows that it binds preferentially to the U4/U6 hybrid snRNAs compared to other snRNAs. The interaction between Prp24 and the U4/U6 hybrid appears to involve two regions in the RNA: the 39-57 region of U6 and stem II of the U4/U6 hybrid. Interestingly, some U4 mutations, which destabilize stem II, increase the affinity of Prp24 for the U4/U6 RNAs compared to the wild type. This suggests that the binding of Prp24 to the U4/U6 RNAs may involve some destabilization of the RNA duplex. We also found that Prp24 can stimulate the annealing of U4 and U6, suggesting that Prp24 participates in both the formation and disassembly of the U4/U6 hybrid during splicing.     

Evidence for a ribonucleoprotein complex as a template for microtubule initiation in vivo

At the onset of mitosis in cultured mammalian cells, the centriolar region rapidly initiates the assembly of microtubules (MT) to form two asters which ultimately forms the basis of the mitotic spindle. The rapid change in the centrosphere's ability to nucleate MTs at prometaphase maybe due to the presence of an RNase sensitive component. Lysis of colcemid-blocked mitotic cells with RNase A or T₂ prior to addition of exogenous microtubule protein greatly diminishes the number of MT that can be nucleated in a lysed cell system. MT initiation can also be reduced or abolished by extended lysis in neutral buffers or brief lysis in acidic buffers. This suggests that the RNA component maybe complexed with a protein to serve as a template for MT initiation at the onset of mitosis.     

Evidence for existence of messenger ribonucleoprotein complexes in Tetrahymena pyriformis

When polysomes from Tetrahymena pyriformis pulse-labeled with ³²P-orthophosphate are dissociated and analysed on sucrose gradients, a large amount of labeled material is found in the 4–23 S region of the gradients.From labeling experiments a half-life of decay of 10.5 min is estimated for the 4–23S material. When cells are pulse-labeled with amino acids, no protein incorporation is found in the 4–23 S material but most of the material is retained on Millipore filters. The sedimentation values of the 4–23 S material are decreased after SDS-treatment. When polysomes from pulse-labeled cells are dissociated and analysed on CsCl-gradients, some rapidly labeled RNP-particles are observed with buoyant densities ranging from 1.51-1.47 g/cm³.     

Evidence for the existence of a structural RNA component in the nuclear ribonucleoprotein particles containing heterogeneous RNA

The structural organisation of nuclear ribonucleoprotein particles carrying the HnRNA has been investigated. Experiments are presented which indicate the existence in the RNP particles of two different RNA species: (1) the rapidly labelled HnRNA and (2) stable, low molecular weight RNA, probably located in the interior of the protein moiety, which may serve a structural role for the arrangement of the proteins within the RNP particle.     

Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.     

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.