Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6

Antibodies specific for N6-methyladenosine (m6A) were elicited in rabbits and used to study the accessibility in intact snRNPs of the m6A residues present in the snRNAs U2, U4 and U6. The antibody quantitatively precipitates snRNPs U2 and U4/U6 from total nucleoplasmic snRNPs U1-U6 isolated from HeLa cells, which demonstrates that the m6A residues of the respective snRNAs are not protected by snRNP proteins in the snRNP particles. While the anti-m6A IgG does not react at all with U5 RNPs lacking m6A, a significant amount of U1 RNPs was co-precipitated despite the fact that U1 RNA does not contain m6A either. Since anti-m6A IgG does not react with purified U1 RNPs and co-precipitation of U1 RNPs is dependent on the presence of U2 RNPs but not of U4/U6 RNPs, these data indicate an interaction between snRNPs U1 and U2 in vitro. The anti-m6A precipitation pattern described above was also observed with snRNPs isolation from mouse Ehrlich ascites tumor cells, indicating similar three-dimensional arrangements of snRNAs in homologous snRNP particles from different organisms.     

The structure of mammalian small nuclear ribonucleoproteins. Identification of multiple protein components reactive with anti-(U1)ribonucleoprotein and anti-Sm autoantibodies

The Sm small nuclear ribonucleoproteins (snRNPs) from mammalian cells have been characterized as containing U1, U2, U4, U5, and U6 RNA associated with some subset of at least 10 distinct polypeptides (called 68K, A, A', B, B', C, D, E, F, and G) that range in molecular weight from 68,000 to 11,000. Whereas this entire collection of snRNP particles is precipitated by patient anti-Sm autoantibodies, anti-(U1)RNP autoantibodies specifically recognize U1 snRNPs. Here, we have performed immunoblots using the sera from 29 patients and a mouse anti-Sm monoclonal antibody to identify which HeLa cell snRNP proteins carry anti-Sm or anti-(U1)RNP antigenic determinants. Strikingly, every serum surveyed, as well as the monoclonal antibody, recognizes determinants on two or more snRNP protein components. The three proteins, 68K, A, and C, that uniquely fractionate with U1 snRNPs are specifically reactive with anti-(U1)RNP sera in blots. Anti-Sm patient sera and the mouse monoclonal antibody react with proteins B, B', D, and sometimes E, one or more of which must be present on all Sm snRNPs. The blot results combined with data obtained from a refined 32P-labeled RNA immunoprecipitation assay reveal that, in our collection of the sera from 29 patients, anti-Sm rarely exists in the absence of equal or higher titers of anti-(U1)RNP; moreover, (U1)RNP sera often contain detectable levels of anti-Sm. Our findings further define the protein composition of the Sm snRNPs and raise intriguing questions concerning the relatedness of snRNP polypeptides and the mechanism of autoantibody induction.     

Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome

Autoantibodies directed against the U2 small nuclear ribonucleoprotein (snRNP) have been found in the serum of a patient with scleroderma-polymyositis overlap syndrome. This specificity, called anti-(U2)-RNP, is distinct from all previously described autoantibodies, including those that precipitate related snRNPs: anti-Sm antibodies, which react with the entire set of U1, U2, U4, U5, and U6 snRNPs, and anti-(U1)RNP antibodies, which recognize only U1 snRNPs. From HeLa cell extracts, anti-(U2)RNP immunoprecipitates predominantly one 32P-labeled RNA species, identified as U2 small nuclear RNA, and six [35S]methionine-labeled protein bands, A' (Mr = 32,000), B (Mr = 28,000), D (Mr = 16,000), E (Mr = 13,000), F (Mr = 12,000), and G (Mr = 11,000). Protein blot analysis reveals that the A' protein carries (U2)RNP antigenic determinant(s) and therefore represents a polypeptide unique to the U2 snRNP; the B protein associated with U2 snRNPs may also be unique. Like U1 and the other Sm snRNPs, U2 snRNPs occupy a nuclear, non-nucleolar location and are antigenically conserved from insects to man. An antibody specific for the U2 snRNP will be useful in deciphering the function of this particle.     

Immunoprecipitation distinguishes non-overlapping groups of snRNPs in Schizosaccharomyces pombe.

The large number of snRNAs in the fission yeast Schizosaccharomyces pombe can be divided into four non-overlapping groups by immunoprecipitation with antibodies directed against mammalian snRNP proteins. 1) Of the abundant snRNAs, anti-Sm sera precipitate only the spliceosomal snRNAs U1, U2, U4, U5 and U6. Surprisingly, three Sm-sera tested distinguish between U2, U4 and U5 and U1 from S.pombe; one precipitating only U1 and two precipitating U2, U4 and U5 but not U1. 2) A group of 11 moderately abundant snRNAs are not detectably precipitated by human anti-Sm sera, but are specifically precipitated by monoclonal antibody H57 specific for the human B/B' polypeptides. From Aspergillus nidulans this antibody also precipitates at least 12 snRNAs. 3) Anti-(U3)RNP sera do not precipitate the above snRNAs, but precipitate at least 6 further snRNAs, including the homologues of U3. Both the anti-(U3)RNP sera and H57 also efficiently precipitate a number of discrete non-capped RNAs. 4) A small number of additional snRNAs are not detectably precipitated by any anti-serum tested to date, further analysis may identify antisera specific for these snRNPs. Western blots of purified snRNP proteins were used to identify the S.pombe proteins responsible for these immunoprecipitations. Several Sm-sera decorate a 16.3kD protein which may be a D protein homologue, monoclonal H57 decorates a further protein of 16kD and an anti-(U3)RNP serum decorates the homologue of the 36kD U3-specific protein, fibrillarin.     

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.     

Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.     

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     

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.     

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.     

RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.     

Characterization of U small nuclear RNA-associated proteins

Differential immunoaffinity chromatography using a combination of autoimmune antibodies allows for the rapid bulk separation of specific small nuclear ribonucleoproteins (snRNPs). Passage of a HeLa cell extract over a column constructed of human anti-Sm autoantibodies results directly in the elution of complexes containing the small nuclear RNA species, U1, U2, U4, U5, and U6, and nine major polypeptides of molecular weight 69,000, 32,000, 27,000, 26,000, 18,500, 13,000, 11,000 doublet, and less than 10,000. Passage of crude extracts through a column bearing murine monoclonal antibodies directed against the 69,000 molecular weight (U1)RNP peptide gives an enriched population of U1 snRNP particles in the retained material. When the flowthrough material from the (U1)RNP column is passed through an anti-Sm column, the retained material is enriched in U2, U4, U5 plus U6 snRNP complex. The 69,000, 32,000, and 18,500 molecular weight polypeptides are confined to the U1 fraction while the remaining proteins are recovered in both fractions. The procedure is simple and rapid, producing complexes with a high degree of resolution and in sufficient yield to provide a ready source of snRNP complexes for functional studies.     

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.     

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.     

Autoantibodies to ribonucleoprotein particles containing U2 small nuclear RNA.

Autoantibodies exclusively precipitating U1 and U2 small nuclear ribonucleoprotein (snRNP) particles [anti-(U1,U2)RNP] were detected in sera from four patients with autoimmune disorders. When tested by immunoblotting, these sera recognized up to four different protein antigens in purified mixtures of U1-U6 RNP particles. With purified antibody fractions eluted from individual antigen bands on nitrocellulose blots, each anti-(U1,U2)RNP serum precipitated U2 RNP by virtue of the recognition of a U2 RNP-specific B" antigen (mol. wt. 28 500). Antibodies to the U2 RNP-specific A' protein (mol. wt. 31 000) were found in only one serum. The B" antigen differs slightly in mol. wt. from the U1-U6 RNA-associated B/B' antigens and can be separated from this doublet by two-dimensional gel electrophoresis, due to its more acidic pI. In immunoprecipitation assays, the purified anti-B" antibody specificity also reacts with U1 RNPs which is due to cross-reactivity of the antibody with the U1 RNA-specific A protein, as demonstrated by immunoblotting using proteins from isolated U1 RNPs as antigenic material. Thus the A antigen not only bears unique antigenic sites for anti-A antibodies contained in anti-(U1)RNP sera, it also shares epitopes with the U2 RNP-specific B" antigen.     

Pre-mRNA splicing by complementation with purified human U1, U2, U4/U6 and U5 snRNPs.

The four major nucleoplasmic small nuclear ribonucleoprotein particles U1, U2, U4/U6 and U5 can be extensively purified from HeLa cells by immunoaffinity chromatography using a monoclonal anti-trimethylguanosine antibody. The snRNP particles in active splicing extracts are selectively bound to the immunoaffinity matrix, and are then gently eluted by competition with an excess of free nucleoside. Biochemical complementation studies show that the purified snRNPs are active in pre-mRNA splicing, but only in the presence of additional non-snRNP protein factors. All the RNPs that are necessary for splicing can be purified in this manner. The active snRNPs are characterized with respect to their polypeptide composition, and shown to be distinct from several other activities implicated in splicing.     

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).     

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

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.     

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts.

RNA duplexes containing the modified base 2-amino-adenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A.U base pairs. Antisense 2'-O-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not U1 and U2 snRNPs with pre-mRNA.