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.     

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.     

Evidence for three distinct D proteins, which react differentially with anti-Sm autoantibodies, in the cores of the major snRNPs U1, U2, U4/U6 and U5.

Electrophoresis of the mixture of proteins from purified snRNPs U1, U2, U4/U6 and U5 on SDS-polyacrylamide gels that had been allowed to polymerise in the presence of high TEMED concentrations have revealed the presence of proteins in the snRNPs that previously had eluded detection. The most striking case is that of protein D, heretofore generally observed as a single broad band; in high-TEMED gels, this splits into three clearly-separated bands, identified as three distinct proteins. We have denoted these proteins D1 (16 kDa), D2 (16.5 kDa) and D3 (18 kDa). Chemical and immunological studies have shown that D1 is identical with the common snRNP protein D, whose structure was recently resolved by cDNA cloning (Rokeach et al. (1988), Proc. Natl. Acad. Sci. USA, 85, 4832-4836) and that D2 and D3 are clearly distinct from D1 and very probably from each other. In addition to D1, proteins D2 and D3 are present in purified U1, U2, U4/U6 and U5 snRNPs isolated from HeLa cells, so these also belong to the group of common snRNP proteins. They are also found in snRNPs isolated from mouse cells, indicating that the role of these proteins in the structure and/or function of UsnRNPs has been conserved in evolution. Interestingly, patients with systemic lupus erythematosus produce populations of anti-Sm autoantibodies that react differentially with the D proteins; some recognise all of them and others only a subset. The high-TEMED gels allow improved resolution not only of the D proteins, but also of some of the U5-specific proteins contained in 20S U5 snRNPs, in particular the 15-kDa protein. In addition, under these conditions, the common G protein, previously observed as a single band, appears as a doublet. Whether the additional band represents a distinct common snRNP protein or a post-translationally modified version of G is not yet known.     

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.     

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.     

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.     

An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis.

The particle state of U snRNPs was analyzed in oocytes, eggs, embryos and testes from Xenopus laevis. In each case both the relative abundance and the composition of some U snRNPs were found to differ from that of somatic cells. U2 and U6 snRNPs were the most prominent U snRNPs in germ cells and early embryos. In particular, the concentration of U6 snRNA was 10-20 times higher than that of U4 snRNA. Most of the U6 snRNA was not associated with U4 snRNA and migrated on sucrose gradients as a U6 snRNP. The structure of this novel U snRNP was analyzed. A single protein of 50 kd was copurified with U6 snRNPs by a combination of gradient fractionation, immunodepletion with anti-Sm antibodies and immunoprecipitation with anti-6-methyl adenosine antibodies. Although the U6 snRNP did not contain Sm proteins it migrated into the nucleus when U6 snRNA was injected into the cytoplasm of oocytes. Two U6 snRNA elements have been identified. The first is essential for nuclear migration in oocytes, but not for the formation of U4/6 snRNPs in vitro and might be the binding site of a U6-specific protein. The second element was required for interaction with U4 snRNPs but not for nuclear targeting.     

Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei.

Polyclonal antibodies were raised against purified protein components of the U2 small nuclear ribonucleoprotein (snRNP) from Trypanosoma brucei. Through immunoblot and immunoprecipitation analyses three antisera were characterized that reacted specifically with U2 snRNP proteins of molecular weights 40,000 (anti-40K) and 16,500 (anti-16.5K), and with each of four proteins of molecular weights 14,000, 12,500, 10,000, and 8,500 (anti-CP). Anti-40K antibodies specifically immunoprecipitated the U2 snRNP from trypanosomal extracts, whereas anti-CP antibodies recognized several snRNPs, including the SL RNP and the U2 and U4/U6 snRNPs; in addition, minor RNAs were detected, suggesting that a family of snRNPs with common or related protein components exists in trypanosomes. None of these antibodies cross-reacted significantly with total mammalian snRNP proteins, indicating that the trypanosomal snRNP proteins are immunologically distinct from their mammalian counterparts. Using immunofluorescence microscopy, the snRNP proteins exhibited a differential cellular distribution. Whereas the 40-kDa protein is localized exclusively in the nucleus, with the nucleolus being excluded, a fraction of the common proteins also resides in the cytoplasm.     

Detection of intranuclear clusters of Sm antigens with monoclonal anti-Sm antibodies by immunoelectron microscopy

It has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti-Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti-Sm antibodies in HeLa cells.     

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.     

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.     

Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents.

A procedure is described for the purification of the individual major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 from HeLa cells. The salient feature of the method is the combined usage of antibodies against 2,2,7-trimethylguanosine (m3G) and 6-methyladenosine (m6A) for differential immune affinity chromatography of the snRNPs. While anti-m3G affinity columns allow the separation of snRNPs U1, U2 and U5 from U4/U6 RNPs, anti-m6A antibodies selectively react with snRNPs U2 and U4/U6. Our technique further incorporates immune affinity chromatography of snRNPs with antibodies against snRNP proteins in addition to ion exchange chromatography. The procedure avoids the usage of denaturing agents, so as to maintain the native structure of the particles. This is mainly provided for by the possibility of eluting the anti-m3G and anti-m6A bound snRNPs with excess of the respective nucleosides. We have so far identified 12 polypeptides as constituents of the major snRNPs U1 to U6. Seven proteins of approximate mol. wts 29 kd (B'), 28 kd (B), 16 kd (D), 15.5 kd (D'), 12 kd (E), 11 kd (F) and 9 kd (G) were present in each of the individual snRNPs U1, U2, U5 and U4/U6. In addition to the common proteins, U1 RNPs contain three unique polypeptides of mol. wts 70 kd, 34 kd (A) and 22 kd (C). U2 RNPs are characterized by the presence of a 33-kd and a 28.5-kd protein, denoted A' and B". We could not detect any unique polypeptide confined to the purified snRNPs U5 or U4/U6.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isofocusing of antigenic small nuclear ribonucleoproteins. II. Preparative isolation

In a companion report (T.B. Okarma, W.S. Schrier, and R. Feinbaum, 1985, Anal. Biochem. 147, 27-37) the behavior of small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels was characterized. This communication extends those findings and describes a gentle procedure for the preparative isolation of snRNPs in native form from cultured murine L-5178y leukemia cells using sucrose density gradient centrifugation, preparative isofocusing, and gel filtration chromatography. Isofocusing in granulated gels separated intact uridylic acid (U)-snRNPs from tRNA and La RNPs. The U-snRNPs remained immunoprecipitable by lupus antisera throughout fractionation. The final product obtained in 2% yield contained primarily U1 and U2 snRNAs and lesser amounts of U3, U4, U5, and U6, along with the core U-snRNP polypeptides A-G. The core polypeptides displayed apparent pI's which ranged from 4.5 to 9.5 when analyzed by two-dimensional gel electrophoresis. Proteins B (28,000), D (16,000), and E (13,000) exhibited isoelectric variants. The Sm determinant proteins B' (28,000) and E (13,000) isofocused as basic peptides with apparent pI's of 9.5 and 8.5, respectively. The purity of the final fractions compared well with that of immunoprecipitates and the procedure reproducibly generated yields of native snRNPs sufficient for in vitro studies of their biological function.     

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.     

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.     

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.     

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.