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)     

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.     

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.     

Analysis of HPV-1 E4 gene expression using epitope-defined antibodies.

Six monoclonal antibodies (mAbs) have been raised against the E4 proteins of HPV-1. Five of these were found to recognize denaturation-resistant epitopes as determined by Western blotting--and their binding sites were identified by determining their reactivity against a panel of bacterial E4--beta-galactosidase fusion proteins which contained progressive deletions at the C-terminal end of the E4 region. The five mAbs were found to bind to four distinct sites. By using these epitope-defined mAbs, along with anti-peptide antibodies raised against putative N- and C-terminal E4 sequences, we have determined the relationships between the eight distinct polypeptides (mol. wt 10/11 kd, 16/17 kd, 21/23 kd and 32/34 kd) previously shown to be expressed from the E4 gene of HPV-1 in productively infected papillomas. The 17 kd E4 polypeptide appears to be the product of a spliced mRNA encoding five amino acids from open reading frame (ORF) E1 joined onto 120 from the E4 ORF. The 16 kd and 10/11 kd proteins, which may be derived from this, lack sequences (approximately 15 and 70 amino acids respectively) encoded by the 5' end of the E4 gene. The 32/34 kd proteins were detected by all antibodies which reacted with the 16/17 kd polypeptides, suggesting that they represent dimers of the latter species. The 21/23 kd polypeptides, however, do not appear to be simple dimers of the 10/11 kd protein as previously predicted, and reacted with antibodies whose epitopes mapped in the N-terminal half of the E4 protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.     

Evolutionary conservation of minor U12-type spliceosome between plants and humans

Splicing of rare, U12-type or AT-AC introns is mediated by a distinct spliceosome that assembles from U11, U12, U4atac, U6atac, and U5 snRNPs. Although in human cells the protein composition of minor and major snRNPs is similar, differences, particularly in U11 and U12 snRNPs, have been recently described. We have identified an Arabidopsis U11 snRNP-specific 35K protein as an interacting partner of an RS-domain-containing cyclophilin. By using a transient expression system in Arabidopsis protoplasts, we show that the 35K protein incorporates into snRNP. Oligo affinity selection and glycerol gradient centrifugation revealed that the Arabidopsis 35K protein is present in monomeric U11 snRNP and in U11/U12-di snRNP. The interaction of the 35K protein with Arabidopsis SR proteins together with its strong sequence similarity to U1-70K suggests that its function in splicing of minor introns is analogous to that of U1-70K. Analysis of Arabidopsis and Oryza sativa genome sequences revealed that all U11/U12-di-snRNP-specific proteins are conserved in dicot and monocot plants. In addition, we have identified an Arabidopsis gene encoding the homolog of U4atac snRNA and a second Arabidopsis gene encoding U6atac snRNA. Secondary structure predictions indicate that the Arabidopsis U4atac is able to form dimeric complexes with both Arabidopsis U6atac snRNAs. As revealed by RNaseA/T1 protection assay, the U4atac snRNA gene is expressed as an ~160-nt RNA, whereas the second U6atac snRNA gene seems to be a pseudogene. Taken together, our data indicate that recognition and splicing of minor, AT-AC introns in plants is highly similar to that in humans.     

Fractionation and characterization of human small nuclear ribonucleoproteins containing U1 and U2 RNAs

Human small nuclear ribonucleoproteins (snRNPs) containing U1 and U2 snRNAs have been isolated from cultured cells by nonimmunological methods. The U1 snRNP population remained immunoprecipitable by systemic lupus erythematosis anti-RNP and anti-Sm antibodies throughout fractionation and contained polypeptides of molecular weights corresponding to those defined as U1 snRNP polypeptides by immunoprecipitation of crude extracts. The purified assemblies contained U1 RNA and nine snRNP polypeptides of molecular weights 67,000 (P67), 30,000 (P30), 23,000 (P23), 21,500 (P22), 17,500 (P18), 12,300 (P12), 10,200 (P10), 9,100 (P9), and 8,500 (P8). P67, P30, and P18 were unique to U1 snRNPs. The U2 snRNP population remained immunoprecipitable by the systemic lupus erythematosis anti-Sm antibody throughout fractionation. The purified U2 assemblies contained six polypeptides of molecular weights corresponding to those defined by immunoprecipitation to be common to U1 and U2 snRNPs including P23, P22, P12, P10, P9, and P8. In addition, U2 snRNPs contained a unique polypeptide of 27,000 Da.     

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.     

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.     

Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis

The distribution of small nuclear ribonucleoprotein particles containing U snRNAs (U snRNPs) during oogenesis and early development in Xenopus was analyzed with a lupus antibody (anti-Sm) that reacts with snRNA-binding proteins. Fully grown oocytes and embryos prior to gastrulation were found to be relatively depleted of U snRNPs in their nuclei and to contain an excess of snRNA-binding proteins stored in the cytoplasm. During late blastula-early gastrula, or after microinjection of U snRNAs into the cytoplasm of a mature oocyte, the proteins migrate into the nucleus. Dot hybridization analysis showed that small previtellogenic oocytes already contain a maximal amount of U1 (and U2) snRNAs, which then decreases to about 20% of that value in fully mature oocytes, even though the cell's volume has increased enormously. Thus fully grown oocytes and eggs accumulate snRNA-binding proteins for use during early development, but this is not coupled with the accumulation of U snRNA.     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.     

Characterization of autoantibodies that recognize U4/U6 small ribonucleoprotein particles in serum from a patient with primary Sj?gren's syndrome.

We identified autoantibodies that recognize the U4/U6 snRNPs in a serum from a 63-year-old Japanese patient (TT) with primary Sj?gren's syndrome. This patient's serum immunoprecipitated U4 and U6 sn-RNAs exclusively from 32P-labeled HeLa cell extracts and a newly identified 120-kDa protein along with the Sm core proteins (B'/B, D, E, F, and G) from [35S] methionine-labeled HeLa cell extracts. Immunoblotting demonstrated that only the 120-kDa protein was recognized by this unique serum. In glycerol density gradient centrifugation, the 120-kDa protein reactive with TT serum cosedimented with U4 and U6 snRNAs, suggesting that the 120-kDa protein is a unique component of the U4/U6 snRNP particle. In the same study, the U4/U6 snRNP precipitated by TT serum sedimented only in the lower density, whereas anti-Sm antibodies precipitated U4/U6 snRNAs in a broad range of the gradient. This result suggests the presence of at least two molecular forms of the U4/U6 snRNP particles; larger particles, probably the U4/U5/U6 snRNP complex, and free particles. Thus, the U4/U6 snRNP recognized by TT serum includes the U4 and U6 snRNAs, with Sm core proteins, and the novel 120-kDa protein, and appears to be a free particle not associated with larger complexes.     

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.     

U1 small nuclear RNA variants differentially form ribonucleoprotein particles in vitro

The U1 small nuclear (sn)RNA participates in splicing of pre-mRNAs by recognizing and binding to 5′ splice sites at exon/intron boundaries. U1 snRNAs associate with 5′ splice sites in the form of ribonucleoprotein particles (snRNPs) that are comprised of the U1 snRNA and 10 core components, including U1A, U1-70K, U1C and the ‘Smith antigen’, or Sm, heptamer. The U1 snRNA is highly conserved across a wide range of taxa; however, a number of reports have identified the presence of expressed U1-like snRNAs in multiple species, including humans. While numerous U1-like molecules have been shown to be expressed, it is unclear whether these variant snRNAs have the capacity to form snRNPs and participate in splicing. The purpose of the present study was to further characterize biochemically the ability of previously identified human U1-like variants to form snRNPs and bind to U1 snRNP proteins. A bioinformatics analysis provided support for the existence of multiple expressed variants. In vitro gel shift assays, competition assays, and immunoprecipitations (IPs) revealed that the variants formed high molecular weight assemblies to varying degrees and associated with core U1 snRNP proteins to a lesser extent than the canonical U1 snRNA. Together, these data suggest that the human U1 snRNA variants analyzed here are unable to efficiently bind U1 snRNP proteins. The current work provides additional biochemical insights into the ability of the variants to assemble into snRNPs.     

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.     

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.