Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs

U₁ snRNP² isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U₁ molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U₁ core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U₁ snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U₁, U₂, U₄, U₅ and U₆ snRNPs, which contains the same four/five polypeptides as U₁ snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U₂, U₄ and U₅ were found in addition to those derived from U₁. No fragment derived from U₆ was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)_nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U₂, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U₆ snRNA, it does not contain the sequence A(U)_nG which correlates well with the fact that no U₆ snRNA fragment seems to resist micrococcal nuclease digestion.     

Mg2+ induces a sharp and reversible transition in U1 and U2 small nuclear ribonucleoprotein configurations.

When U1 and U2 small nuclear ribonucleoproteins (snRNPs) purified by a procedure which preserves their immunoprecipitability by autoimmune antibodies (Hinterberger et al., J. Biol. Chem. 258:2604-2613, 1983), were submitted to extensive digestion with micrococcal nuclease, we found that their degradation pattern was sharply dependent upon magnesium concentration, indicating that they undergo a profound structural modification. At low Mg2+ (less than or equal to 5 mM), both particles only exhibit a core-resistant structure previously identified as being common to all but U6 snRNAs (Liautard et al., J. Mol. Biol. 162: 623-643, 1982). At high Mg2+ (greater than or equal to 7 mM), U1 and U2 snRNPs behave differently from one another. In U1 snRNP, most U1 snRNA sequence is protected, except for the 10 5'-terminal nucleotides presumably involved in splicing and a short sequence between nucleotides 102 and 108. Another region spanning nucleotides 60 to 79 is only weakly protected. This structural modification was demonstrated to be reversible. In U2 snRNP, the U2 snRNA sequence remains exposed in its 5' part up to nucleotide 92, and the 3'-terminal hairpin located outside the core structure becomes protected.     

Conserved loop I of U5 small nuclear RNA is dispensable for both catalytic steps of pre-mRNA splicing in HeLa nuclear extracts

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.     

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.     

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.     

Protein-RNA interactions in 20S U5 snRNPs.

The interaction of the U5-specific polypeptides with U5 snRNA was investigated by comparison of the differential accessibility towards nucleases and dimethylsulfate of defined regions of U5 snRNA in purified 20S and 10S U5 snRNPs. While 20S U5 snRNPs contain eight U5-specific proteins in addition to the common proteins, the 10S U5 snRNPs contain only the latter proteins. The results indicate that only the central part of stem/loop I of U5 snRNA including internal loops IL2 and IL2', contains binding sites for U5-specific proteins, suggesting that several U5-specific proteins may be bound to U5 snRNP via protein-protein interactions. Moreover, they show that the core polypeptides do not interact with stem/loop I.     

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

An in vitro reconstitution/splicing complementation system has been developed which has allowed the investigation of the role of mammalian U2 and U5 snRNP components in splicing. U2 or U5 snRNP cores are first reconstituted from purified native snRNP core proteins and snRNA in the absence of cellular extract and are subsequently added to splicing extracts depleted of either U2 or U5 snRNP. When snRNPs reconstituted with HeLa U2 or U5 snRNA were added to U2- or U5-depleted nuclear extract, splicing was complemented. Addition of naked snRNA, on the other hand, did not restore splicing, demonstrating that the core proteins are essential for both U2 and U5 snRNP functions in splicing. Hybrid U2 or U5 snRNPs, reconstituted with core proteins isolated from U1 or U2 snRNPs, were equally active in splicing complementation, indicating that the snRNP core proteins are functionally interchangeable. U5 snRNPs reconstituted from in vitro transcribed U5 snRNA restored splicing to a level identical to that observed with particles reconstituted from authentic HeLa U5 snRNA. In contrast, splicing could not be restored to U2-depleted extract by the addition of snRNPs reconstituted from synthetic U2 snRNA, suggesting that U2 snRNA base modifications are essential for U2 snRNP function.     

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.     

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.     

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.     

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.     

Characterization of U6 snRNA-protein interactions

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3' end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3' half of U6, which contains the 3' stem-loop and uridine-rich 3' end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.     

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.     

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera

Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.     

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster

The U7 snRNP involved in histone RNA 3' end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs.     

Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assembly and splicing by snRNP-free nuclear proteins.

As demonstrated by RNase T1 protection assays at 0 degrees C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloride bind to the 5' and 3' splice sites of human beta-globin pre-mRNA, respectively. We also devised a saturation-complementation assay and have found that this purified U5 snRNP, unlike U1, successfully competes with snRNP-free fractions of nuclear proteins which inhibit spliceosome assembly and splicing. Restoration of activity requires intact U5 snRNA and correlates with the presence of the 100 Kd intron binding protein (IBP) which we have previously characterized (Tazi et al., 1986, Cell 47, 755-766). Our results are compatible with a model in which the recognition of the 3' splice site by IBP-U5 snRNP is one of the earliest events of the spliceosome assembly. It could organize the structure of the 3' splice site region of the human beta-globin like pre-mRNAs. However, on the basis of results showing that beta-globin and major late adenovirus seem to have different requirements with respect to IBP-U5 snRNP, it appears that some pre-mRNAs could have a native structure that necessitates less if at all IBP-U5.     

In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA

In this paper we describe a method for preparing native, RNA-free, proteins from anti-m₃G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B, D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm³. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.Abbreviations PEG Polyethelene glycol - PMSF Phenylmethyl sulfonylfluoride - TP total proteins - mAb monoclonal antibody     

p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor

During each spliceosome cycle, the U6 snRNA undergoes extensive structural rearrangements, alternating between singular, U4-U6 and U6-U2 base-paired forms. In Saccharomyces cerevisiae, Prp24 functions as an snRNP recycling factor, reannealing U4 and U6 snRNAs. By database searching, we have identified a Prp24-related human protein previously described as p110(nrb) or SART3. p110 contains in its C-terminal region two RNA recognition motifs (RRMs). The N-terminal two-thirds of p110, for which there is no counterpart in the S.cerevisiae Prp24, carries seven tetratricopeptide repeat (TPR) domains. p110 homologs sharing the same domain structure also exist in several other eukaryotes. p110 is associated with the mammalian U6 and U4/U6 snRNPs, but not with U4/U5/U6 tri-snRNPs nor with spliceosomes. Recom binant p110 binds in vitro specifically to human U6 snRNA, requiring an internal U6 region. Using an in vitro recycling assay, we demonstrate that p110 functions in the reassembly of the U4/U6 snRNP. In summary, p110 represents the human ortholog of Prp24, and associates only transiently with U6 and U4/U6 snRNPs during the recycling phase of the spliceosome cycle.     

The PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5'' portion of the U4 small nuclear RNA.

We have combined oligonucleotide-directed RNase H degradation and immunoprecipitation in a study of the association of the Saccharomyces cerevisiae PRP4 protein with the U4-U6 complex. We have found that three oligonucleotides were able to direct nearly to completion the RNase H-specific cleavage of the target RNA molecules as they exist in splicing extracts. Immunoprecipitation of the degradation products with PRP4 antibody showed that the 5' portion of U4 small nuclear RNA (snRNA) and the 3' portion of U6 snRNA coimmunoprecipitated with the PRP4 protein. Micrococcal nuclease protection experiments confirmed further that the 5' portion and 3' end of U4 snRNA were very resistant to nuclease digestion, whereas the 3' portion of U6 snRNA was protected to only a very small extent. We conclude that the PRP4 protein of S. cerevisiae is associated primarily with the 5' portion of U4 snRNA in the U4-U6 small nuclear ribonucleoprotein (snRNP).