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.     

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.     

An element in human U6 RNA destabilizes the U4/U6 spliceosomal RNA complex.

Large-scale changes in RNA secondary structure, such as those that occur in some of the spliceosomal RNAs during pre-mRNA splicing, have been proposed to be catalyzed by ATP-dependent RNA helicases. Here we show that deproteinized human U4/U6 spliceosomal RNA complex, which has the potential for extensive intermolecular base pairing, contains a cis-acting element that promotes its dissociation into free U4 and U6 RNAs. The destabilzing element corresponds to the bae of putative intramolecular stem in U6 RNA that includes the 3' three-quarters of the molecule. Oligonucleotides expected to compete for U6 RNA 3' stem formation promote assembly of the human U4/U6 RNA complex under conditions that otherwise result in dissociation of the U4/U6 complex. Truncation of the putative 3' stem-forming sequences in U6 RNA by oligonucleotide-directed RNase H cleavage increases the melting temperature of the U4/U6 RNA complex by almost 20 degree C, to a level commensurate with its intermolecular base-pairing potential. We conclude that the stability of the competing human U6 RNA intramolecular 3' stem, combined with a low activation energy for conformational rearrangement, causes the human U4/U6 RNA complex to be intrinsically unstable despite its base-pairing potential. Therefore a helicase activity may not be necessary for disassembly of the human U4/U6 complex during activation of the spliceosome. We propose that a previously identified base-pairing interaction between U6 and U2 RNAs may stabilize the human U4/U6 RNA complex by antagonizing U6 RNA 3' stem formation.     

U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.     

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.     

Cross-linking of U2 snRNA using nitrogen mustard. Evidence for higher order structure.

Nuclear mRNA precursors are spliced by a large macromolecular complex called the spliceosome which contains, in most eucaryotes, five small nuclear RNAs (snRNAs) each in the form of a small ribonucleoprotein particle (the U1, U2, U5, and U4/U6 snRNPs). Although secondary structures have been derived for all five spliceosomal snRNAs based on phylogenetic, biochemical, and genetic data, little tertiary structure information is available. Here we use the general cross-linking reagent nitrogen mustard [bis-(2-chloroethyl)methylamine] to detect tertiary interactions within U2 snRNA. After the cross-linking of deproteinized HeLa nuclear extract, two intramolecularly cross-linked U2 species with anomalous electrophoretic mobility can be detected (X-U2#1 and X-U2#2). The 3' and 5' boundaries of each cross-link were determined by rapid enzymatic RNA sequencing of end-labeled RNA. X-U2#1 is cross-linked between the region U41-U55 and G105 or G106, X-U2#2 between U53 and G97 or G98. We then tested the ability of the two cross-linked species to bind snRNP proteins in vitro (in nuclear extract or S100) and in vivo (in Xenopus oocytes). X-U2#2 reconstituted efficiently both in vitro and in vivo but X-U2#1 did not, as judged by immunoprecipitation with antibodies specific for Sm- and U2-specific proteins. Since the cross-link in X-U2#2 involves the Sm binding site but does not block snRNP assembly, our data strongly suggest that the Sm binding site lies on the surface of the native snRNP.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

Ribonucleoprotein organization of eukaryotic RNA. XXX. Evidence that U1 small nuclear RNA is a ribonucleoprotein when base-paired with pre-messenger RNA in vivo

U1 small nuclear RNA is thought to be involved in messenger RNA splicing by binding to complementary sequences in pre-mRNA. We have investigated intermolecular base-pairing between pre-mRNA (hnRNA) and U1 small nuclear RNA by psoralen crosslinking in situ, with emphasis on ribonucleoprotein structure. HeLa cells were pulse-labeled with [3H]uridine under conditions in which hnRNA is preferentially labeled. Isolated nuclei were treated with aminomethyltrioxsalen , which produces interstrand crosslinks at sites of base-pairing between hnRNA and U1 RNA. hnRNA-ribonucleoprotein (hnRNP) particles were isolated in sucrose gradients containing 50% formamide, to dissociate non-crosslinked U1 RNA, and then analyzed by immunoaffinity chromatography using a human autoantibody that is specific for the ribonucleoprotein form of U1 RNA (anti-U1 RNP). After psoralen crosslinking, pulse-labeled hnRNA in hnRNP particles reproducibly bound to anti-U1 RNP. The amount of hnRNA bound to anti-U1 RNP was reduced 80 to 85% when psoralen crosslinking of nuclei was omitted, or if the crosslinks between U1 RNA and hnRNA were photo-reversed prior to immunoaffinity chromatography. Analysis of the proteins bound to anti-U1 RNP after crosslink reversal revealed polypeptides having molecular weights similar to those previously described for U1 RNP. These proteins did not bind to control, non-immune human immunoglobulin G. These results indicate that the subset of nuclear U1 RNA that is base-paired with hnRNA at a given time in the cell is a ribonucleoprotein. This raises the possibility that these proteins, as well as U1 RNA itself, may participate in pre-mRNA splice site recognition by U1 RNP.     

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.     

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethyl-guanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Precursors of U4 small nuclear RNA

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.     

Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.     

Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis.

We found serum autoantibodies directed against the proteins binding exclusively to U4/U6 of Sm small nuclear ribonucleoprotein particle (snRNP) in serum from a patient (MaS) with systemic sclerosis. Their specificity, called anti-MaS, is distinct from that of known antibodies against U snRNP. The U4 and U6 small nuclear RNA from a 32P-labeled HeLa cell extract and five proteins with Mr 150,000, 120,000, 80,000, 36,000, and 34,000, in addition to Sm core proteins (B, B', D, E, F, and G) from an [35S] methionine-labeled extract, were immunoprecipitated by anti-MaS in isotonic solution. However, the Sm core proteins and U4 and U6 small nuclear RNA were separated from the protein-A-Sepharose facilitated MaS immunoprecipitate by incubation in a solution containing 500 mM NaCl. In immunoblots, anti-MaS antibodies reacted with one protein of Mr 150,000 from a HeLa cell nuclear extract that was fractionated by SDS-PAGE and transferred to a nitrocellulose sheet. The monospecific immunoaffinity purified antibody eluted from the immunoblot band immunoprecipitated U4 and U6 small nuclear RNA and reblotted the protein with Mr 150,000. These data indicate that anti-MaS antibodies recognize at least one antigenic protein that binds exclusively to the U4/U6 snRNP.     

U2-U6 RNA folding reveals a group II intron-like domain and a four-helix junction

Intron removal in nuclear precursor mRNA is catalyzed through two transesterification reactions by a multi-megaDalton ribonucleoprotein machine called the spliceosome. A complex between U2 and U6 small nuclear RNAs is a core component of the spliceosome. Here we present an NMR structural analysis of a protein-free U2-U6 complex from Saccharomyces cerevisiae. The observed folding of the U2-U6 complex is a four-helix junction, in which the catalytically important AGC triad base-pairs only within U6 and not with U2. The base-pairing of the AGC triad extends the U6 intramolecular stem-loop (U6 ISL), and the NMR structure of this extended U6 ISL reveals structural similarities with domain 5 of group II self-splicing introns. The observed conformation of the four-helix junction could be relevant to the first, but not the second, step of splicing and may help to position the U6 ISL adjacent to the 5' splice site.     

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.     

In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3' stem-loop

U4 small nuclear RNA (snRNA) plays a fundamental role in the process of premessenger RNA splicing, yet many questions remain regarding the location, interactions, and roles of its functional domains. To address some of these questions, we developed the first in vitro reconstitution system for yeast U4 small nuclear ribonucleoproteins (snRNPs). We used this system to examine the functional domains of U4 by measuring reconstitution of splicing, U4/U6 base-pairing, and triple-snRNP formation. In contrast to previous work in human extracts and Xenopus oocytes, we found that the 3' stem-loop of U4 is necessary for efficient base-pairing with U6. In particular, the loop is sensitive to changes in both length and sequence. Intriguingly, a number of mutations that we tested resulted in more stable interactions with U6 than wild-type U4. Nevertheless, each of these mutants was impaired in its ability to support splicing, indicating that these regions of U4 have functions subsequent to base pair formation with U6. Our data suggest that one such function is likely to be in tri-snRNP formation, when U5 joins the U4/U6 di-snRNP. We have identified two regions, the upper stem of the 3' stem-loop and the central domain, that promote tri-snRNP formation. In addition, the loop of the 3' stem-loop promotes di-snRNP formation, while the central domain and the 3'-terminal domain appear to antagonize di-snRNP formation.