A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic activation (designated BDeltaU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BDeltaU1 and activated B* spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BDeltaU1 spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation. Indeed, immunodepletion/complementation experiments demonstrated that a subset of 35S U5 proteins including Prp19, which form a stable heteromeric complex, are required prior to catalytic step 1 of splicing, but not for stable integration of U4/U6.U5 tri-snRNPs. Thus, comparison of the proteomes of spliceosomal complexes at defined stages can provide information as to which proteins function as a group at a particular step of splicing.     

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.     

The N-terminus of Prp1 (Prp6/U5-102 K) is essential for spliceosome activation in vivo

The spliceosomal protein Prp1 (Prp6/U5-102 K) is necessary for the integrity of pre-catalytic spliceosomal complexes. We have identified a novel regulatory function for Prp1. Expression of mutations in the N-terminus of Prp1 leads to the accumulation of pre-catalytic spliceosomal complexes containing the five snRNAs U1, U2, U5 and U4/U6 and pre-mRNAs. The mutations in the N-terminus, which prevent splicing to occur, include in vitro and in vivo identified phosphorylation sites of Prp4 kinase. These sites are highly conserved in the human ortholog U5-102 K. The results presented here demonstrate that structural integrity of the N-terminus is required to mediate a splicing event, but is not necessary for the assembly of spliceosomes.     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.     

Whole-genome sequencing of a laboratory-evolved yeast strain

Background

Two categories of introns are known, a common U2 type and a rare U12 type. These two types of introns are removed by distinct spliceosomes. The phylogenetic distribution of spliceosomal RNAs that are characteristic of the U12 spliceosome, i.e. the U11, U12, U4atac and U6atac RNAs, suggest that U12 spliceosomes were lost in many phylogenetic groups. We have now examined the distribution of U2 and U12 introns in many of these groups.

Results

U2 and U12 introns were predicted by making use of available EST and genomic sequences. The results show that in species or branches where U12 spliceosomal components are missing, also U12 type of introns are lacking. Examples are the choanoflagellate Monosiga brevicollis, Entamoeba histolytica, green algae, diatoms, and the fungal lineage Basidiomycota. Furthermore, whereas U12 splicing does not occur in Caenorhabditis elegans, U12 introns as well as U12 snRNAs are present in Trichinella spiralis, which is deeply branching in the nematode tree. A comparison of homologous genes in T. spiralis and C. elegans revealed different mechanisms whereby U12 introns were lost.

Conclusions

The phylogenetic distribution of U12 introns and spliceosomal RNAs give further support to an early origin of U12 dependent splicing. In addition, this distribution identifies a large number of instances during eukaryotic evolution where such splicing was lost.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

Comprehensive in vivo RNA-binding site analyses reveal a role of Prp8 in spliceosomal assembly

Prp8 stands out among hundreds of splicing factors as a protein that is intimately involved in spliceosomal activation and the catalytic reaction. Here, we present the first comprehensive in vivo RNA footprints for Prp8 in budding yeast obtained using CLIP (cross-linking and immunoprecipitation)/CRAC (cross-linking and analyses of cDNAs) and next-generation DNA sequencing. These footprints encompass known direct Prp8-binding sites on U5, U6 snRNA and intron-containing pre-mRNAs identified using site-directed cross-linking with in vitro assembled small nuclear ribonucleoproteins (snRNPs) or spliceosome. Furthermore, our results revealed novel Prp8-binding sites on U1 and U2 snRNAs. We demonstrate that Prp8 directly cross-links with U2, U5 and U6 snRNAs and pre-mRNA in purified activated spliceosomes, placing Prp8 in position to bring the components of the active site together. In addition, disruption of the Prp8 and U1 snRNA interaction reduces tri-snRNP level in the spliceosome, suggesting a previously unknown role of Prp8 in spliceosomal assembly through its interaction with U1 snRNA.     

Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3

We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein–protein interactions.     

Two spliceosomes can form simultaneously and independently on synthetic double-intron messenger RNA precursors.

We have investigated the formation of splicing complexes in vitro on mRNA precursors (pre-mRNAs) containing two introns. Sucrose gradient sedimentation analysis revealed that the double-intron substrate becomes associated with 60S structures, which are larger than the 50S splicing complexes we previously observed with single-intron pre-mRNA precursors. We have demonstrated that the 60S complex represents the assembly of two single splicing complexes on the individual introns by conversion of the 60S double splicing complexes into single 50S spliceosomes by oligodeoxynucleotide directed RNase H cleavage of the double-intron pre-mRNAs within the middle exon. In addition, we have observed by native gel electrophoresis a transient double 'pre-splicing' complex analogous to the 35S 'pre-splicing' complex previously found with single-intron pre-mRNAs. Our results indicate that splicing complexes can form independently and simultaneously on the individual introns of multi-intron pre-mRNAs and that the assembly of these multiple spliceosomes proceeds with the same stepwise pathway observed for single-intron RNAs.     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs in the splicing of pre-mRNA molecules. For the preparation of snRNP-depleted nuclear extracts we have first removed the majority of endogenous snRNPs from the nuclear extracts by one passage over an anti-m3G column and then degraded the remaining snRNPs with micrococcal nuclease. The mixture of snRNPs U1, U2, U4/U6 and U5, obtained by anti-m3G immuno-affinity chromatography, was functionally active and able to restore the splicing of snRNP-depleted nuclear extracts. Mono-Q chromatography was used for further fractionation of the snRNPs U1-U6. This produced three fractions that were highly enriched in snRNPs U1 and U2, U5 and U4/U6 respectively. Conditions were found where addition of the [U1, U2] and the U4/U6 snRNP fractions to the snRNP-depleted nuclear extracts gave rise to the formation of splice intermediates in the absence of any 3' cleavage/exon 1-exon 2 product formation. Only when purified 20S U5 snRNPs were added did both steps of the splicing reaction occur efficiently. Our data suggest that U5 snRNP is absolutely required for the second step of splicing and is needed further for efficient initiation of the splicing reaction. The requirement for U5 snRNPs for splicing was corroborated by glycerol gradient sedimentation analysis of the respective reconstituted pre-mRNP complexes. Stable and efficient formation of 50-60S spliceosomes was observed only in the presence of all snRNPs.     

Yeast ntr1/spp382 mediates prp43 function in postspliceosomes

The Ntr1 and Ntr2 proteins of Saccharomyces cerevisiae have been reported to interact with proteins involved in pre-mRNA splicing, but their roles in the splicing process are unknown. We show here that they associate with a postsplicing complex containing the excised intron and the spliceosomal U2, U5, and U6 snRNAs, supporting a link with a late stage in the pre-mRNA splicing process. Extract from cells that had been metabolically depleted of Ntr1 has low splicing activity and accumulates the excised intron. Also, the level of U4/U6 di-snRNP is increased but those of the free U5 and U6 snRNPs are decreased in Ntr1-depleted extract, and increased levels of U2 and decreased levels of U4 are found associated with the U5 snRNP protein Prp8. These results suggest a requirement for Ntr1 for turnover of the excised intron complex and recycling of snRNPs. Ntr1 interacts directly or indirectly with the intron release factor Prp43 and is required for its association with the excised intron. We propose that Ntr1 promotes release of excised introns from splicing complexes by acting as a spliceosome receptor or RNA-targeting factor for Prp43, possibly assisted by the Ntr2 protein.     

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.     

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique.     

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 assembly of a spliceosomal small nuclear ribonucleoprotein particle

The U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) are essential elements of the spliceosome, the enzyme that catalyzes the excision of introns and the ligation of exons to form a mature mRNA. Since their discovery over a quarter century ago, the structure, assembly and function of spliceosomal snRNPs have been extensively studied. Accordingly, the functions of splicing snRNPs and the role of various nuclear organelles, such as Cajal bodies (CBs), in their nuclear maturation phase have already been excellently reviewed elsewhere. The aim of this review is, then, to briefly outline the structure of snRNPs and to synthesize new and exciting developments in the snRNP biogenesis pathways.     

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.     

Saccharomyces cerevisiae NineTeen complex (NTC)-associated factor Bud31/Ycr063w assembles on precatalytic spliceosomes and improves first and second step pre-mRNA splicing efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a ～25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.