U1-specific protein C needed for efficient complex formation of U1 snRNP with a 5' splice site

One of the functions of U1 small nuclear ribonucleoprotein (snRNP) in the splicing reaction of pre-mRNA molecules is the recognition of the 5' splice site. U1 snRNP proteins as well as base-pair interactions between U1 snRNA and the 5' splice site are important for the formation of the snRNP-pre-mRNA complex. To determine which proteins are needed for complex formation, the ability of U1 snRNPs gradually depleted of the U1-specific proteins C, A, and 70k to bind to an RNA molecule containing a 5' splice site sequence was studied in a nitrocellulose filter binding assay. The most significant effect was always observed when protein C was removed, either alone or together with other U1-specific proteins; the binding was reduced by 50 to 60%. Complementation of protein C-deficient U1 snRNPs with purified C protein restored their 5' splice site binding activity. These data suggest that protein C may potentiate the base-pair interaction between U1 RNA and the 5' splice site.     

The Prp19p-associated complex in spliceosome activation

During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.     

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.     

Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.     

Splicing of messenger RNA precursors

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.     

A subset of yeast snRNA's contains functional binding sites for the highly conserved Sm antigen

Autoimmune sera of the Sm specificity react with the major class of small nuclear RNA (snRNA)-containing ribonucleoprotein particles (snRNP's) from organisms as evolutionarily divergent as insects and dinoflagellates but have been reported not to recognize snRNP's from yeast. The Sm antigen is thought to bind to a conserved snRNA motif that includes the sequence A(U3-6)G. The hypothesis was tested that yeast also contains functional analogues of Sm snRNA's, but that the Sm binding site in the RNA is more strictly conserved than the Sm antigenic determinant. After microinjection of labeled yeast snRNA's into Xenopus eggs or oocytes, two snRNA's from Saccharomyces cerevisiae become strongly immunoprecipitable with human auto-antibodies known as anti-Sm. These each contain the sequence A(U5-6)G, are essential for viability, and are constituents of the spliceosome. At least six other yeast snRNA's do not become immunoprecipitable and lack this sequence; these non-Sm snRNA's are all dispensable.     

Intron removal requires proofreading of U2AF/3' splice site recognition by DEK

Discrimination between splice sites and similar, nonsplice sequences is essential for correct intron removal and messenger RNA formation in eukaryotes. The 65- and 35-kD subunits of the splicing factor U2AF, U2AF65 and U2AF35, recognize, respectively, the pyrimidine-rich tract and the conserved terminal AG present at metazoan 3' splice sites. We report that DEK, a chromatin- and RNA-associated protein mutated or overexpressed in certain cancers, enforces 3' splice site discrimination by U2AF. DEK phosphorylated at serines 19 and 32 associates with U2AF35, facilitates the U2AF35-AG interaction and prevents binding of U2AF65 to pyrimidine tracts not followed by AG. DEK and its phosphorylation are required for intron removal, but not for splicing complex assembly, which indicates that proofreading of early 3' splice site recognition influences catalytic activation of the spliceosome.     

Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle.

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.     

Virus-specific splicing inhibitor in extracts from cells infected with HIV-1

Human immunodeficiency virus type 1 (HIV-1), in contrast with most other retroviruses, encodes trans-regulatory proteins for virus gene expression. It is shown in this study, by means of an in vitro splicing system, that nuclear extracts obtained from cells infected with HIV-1 contain a factor (or factors) that specifically inhibits splicing of a synthetic SP6/HIV pre-messenger RNA (pre-mRNA)-containing donor and acceptor splice sites in the coding region for the envelope protein. It is also shown that the SP6/HIV pre-mRNA is not capable of assembly in a ribonucleoprotein complex, spliceosome, in extracts from infected cells. These findings raise the possibility that specific inhibition of pre-mRNA splicing in the envelope protein coding region by HIV-1 trans-regulatory factors might be one control mechanism for efficient production of structural viral proteins and virion assembly.     

An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus

The major small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4 + U6, and U5 have to be transported from the cytoplasm, where they are synthesized, to the nucleus, where they splice pre-messenger RNAs. Since the free core snRNP proteins in the cytoplasm do not enter the nucleus on their own, the nuclear location signal must either reside on the snRNA or be created as a result of snRNA-protein interaction. Here the involvement by the 5'-terminal cap of snRNA molecules in the nucleo-cytoplasmic transport of UsnRNPs has been studied by microinjection of synthetic U1 RNA molecules into frog oocytes; the U1 RNA bore either the normal cap (m3G) or a chemical derivative. Antibodies in the cytoplasm against the m3G cap inhibited the nuclear uptake of U1 snRNP. U1 RNA that was uncapped or contained an unnatural ApppG cap did not enter the nucleus, even though it carried a normal complement of protein molecules. When the ribose ring of the m3G cap was oxidized with periodate, nuclear transport of U1 snRNPs was severely inhibited. Finally, microinjection of m3G cap alone (but not m7G cap) into oocytes severely inhibited the transport of U1 snRNPs to the nucleus. These data suggest that one step in the nuclear uptake of U1 snRNPs involves the m3G cap structure.     

Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome

The splicing process, which removes intervening sequences from messenger RNA (mRNA) precursors is essential to gene expression in eukaryotic cells. This site-specific process requires precise sequence recognition at the boundaries of an intervening sequence, but the mechanism of this recognition is not understood. The splicing of mRNA precursors occurs in a multicomponent complex termed the spliceosome. Such an assembly of components is likely to play a key role in specifying those sequences to be spliced. In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants. This approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction. A minimum of three subunits: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles were identified in the 35S spliceosome structure, which also contains the bipartite RNA intermediate of splicing. A 25S presplicing complex contained only the U2 particle. The multiple subunit structure of the spliceosome has implications for the regulation of a splicing event and for its possible catalysis by ribozyme or ribozymes.     

Heterogeneous nuclear ribonucleoproteins: role in RNA splicing

Splicing in vitro of a messenger RNA (mRNA) precursor (pre-mRNA) is inhibited by a monoclonal antibody to the C proteins (anti-C) of the heterogeneous nuclear RNA (hnRNA)-ribonucleoprotein (hnRNP) particles. This antibody, 4F4, inhibits an early step of the reaction: cleavage at the 3' end of the upstream exon and the formation of the intron lariat. In contrast, boiled 4F4, or a different monoclonal antibody (designated 2B12) to the C proteins, or antibodies to other hnRNP proteins (120 and 68 kilodaltons) and nonimmune mouse antibodies have no inhibitory effect. The 4F4 antibody does not prevent the adenosine triphosphate-dependent formation of a 60S splicing complex (spliceosome). Furthermore, the 60S splicing complex contains C proteins, and it can be immunoprecipitated with 4F4. Depletion of C proteins from the splicing extract by immunoadsorption with either of the two monoclonal antibodies to the C proteins (4F4 or 2B12) results in the loss of splicing activity, whereas mock-depletion with nonimmune mouse antibodies bodies has no effect. A 60S splicing complex does not form in a C protein-depleted nuclear extract. These results indicate an essential role for proteins of the hnRNP complex in the splicing of mRNA precursors.     

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.     

Association of TALS developmental disorder with defect in minor splicing component U4atac snRNA

The spliceosome, a ribonucleoprotein complex that includes proteins and small nuclear RNAs (snRNAs), catalyzes RNA splicing through intron excision and exon ligation to produce mature messenger RNAs, which, in turn serve as templates for protein translation. We identified four point mutations in the U4atac snRNA component of the minor spliceosome in patients with brain and bone malformations and unexplained postnatal death [microcephalic osteodysplastic primordial dwarfism type 1 (MOPD 1) or Taybi-Linder syndrome (TALS); Mendelian Inheritance in Man ID no. 210710]. Expression of a subgroup of genes, possibly linked to the disease phenotype, and minor intron splicing were affected in cell lines derived from TALS patients. Our findings demonstrate a crucial role of the minor spliceosome component U4atac snRNA in early human development and postnatal survival.     

Both catalytic steps of nuclear pre-mRNA splicing are reversible

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.     

The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.     

Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Molecular architecture of the multiprotein splicing factor SF3b

The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined the three-dimensional structure of the human SF3b complex by single-particle electron cryomicroscopy at a resolution of less than 10 angstroms, allowing identification of protein domains with known structural folds. The best fit of a modeled RNA-recognition motif indicates that the protein p14 is located in the central cavity of the complex. The 22 tandem helical repeats of the protein SF3b155 are located in the outer shell of the complex enclosing p14.     

Saccharomyces cerevisiae has a U1-like small nuclear RNA with unexpected properties

Previous experiments indicated that only a small subset of the approximately equal to 24 small nuclear RNAs (snRNAs) in Saccharomyces cerevisiae have binding sites for the Sm antigen, a hallmark of metazoan small nuclear ribonucleoproteins (snRNPs) involved in pre-messenger RNA splicing. Antibodies from human serum to Sm proteins were used to show that four snRNAs (snR7, snR14, snR19, and snR20) can be immunoprecipitated from yeast extracts. Three of these four, snR7, snR14, and snR20, have been shown to be analogs of mammalian U5, U4, and U2, respectively. Several regions of significant homology to U1 (164 nucleotides) have now been found in cloned and sequenced snR19 (568 nucleotides). These include ten out of ten matches to the 5' end of U1, the site known to interact with the 5' splice site of mammalian introns. Surprisingly, the precise conservation of this sequence precludes perfect complementarity between snR19 and the invariant yeast 5' junction (GTATGT), which differs from the mammalian consensus at the fourth position (GTPuAGT).