The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

The RNA helicase-like splicing factor PRP2 interacts only transiently with spliceosomes. To facilitate analysis of interactions of PRP2 with spliceosomal components, PRP2 protein was stalled in splicing complexes by two different methods. A dominant negative mutant form of PRP2 protein, which associates stably with spliceosomes, was found to interact directly with pre-mRNAs, as demonstrated by UV-crosslinking experiments. The use of various mutant and truncated pre-mRNAs revealed that this interaction requires a spliceable pre-mRNA and an assembled spliceosome; a 3' splice site is not required. To extend these observations to the wild-type PRP2 protein, spliceosomes were depleted of ATP; PRP2 protein interacts with pre-mRNA in these spliceosomes in an ATP-independent fashion. Comparison of RNA binding by PRP2 protein in the presence of ATP or gamma S-ATP showed that ATP hydrolysis rather than mere ATP binding is required to release PRP2 protein from pre-mRNA. As PRP2 is an RNA-stimulated ATPase, these experiments strongly suggest that the pre-mRNA is the native co-factor stimulating ATP hydrolysis by PRP2 protein in spliceosomes. Since PRP2 is a putative RNA helicase, we propose that the pre-mRNA is the target of RNA displacement activity of PRP2 protein, promoting the first step of splicing.     

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.     

Roles of PRP8 protein in the assembly of splicing complexes.

Three different approaches have been used to investigate the roles of the yeast U5 snRNP protein PRP8 in spliceosome assembly: genetic depletion of PRP8 protein in vivo, heat inactivation of temperature-sensitive prp8 protein in protoplasts and inhibition of PRP8 function with antibodies in vitro. In each case, U5 and U4/U6 snRNPs failed to assemble into the forming spliceosomes. In addition, extract prepared from PRP8-depleted cells and extract containing inactivated PRP8 protein had substantially reduced amounts of U4/U6.U5 triple snRNP complexes. Thus, functional PRP8 protein is required for the stable formation of U4/U6.U5 complexes without which spliceosomes fail to form. As spliceosome formation was also blocked by anti-PRP8 antibodies that apparently do not disrupt triple snRNPs, PRP8 protein may play a separate role in the assembly of triple snRNPs into spliceosomes. As a consequence of PRP8 depletion the levels of the U4, U5 and U6 snRNAs declined dramatically. We discuss this in the context of the known genetic interactions between PRP8 and putative RNA helicase (DEAD box protein) genes and propose that PRP8 protein plays a role in regulating dynamic RNA-RNA interactions in spliceosome assembly, possibly ensuring the correct directionality of these events.     

Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation.

The elaborate and energy-intensive spliceosome assembly pathway belies the seemingly simple chemistry of pre-mRNA splicing. Prp38p was previously identified as a protein required in vivo and in vitro for the first pre-mRNA cleavage reaction catalyzed by the spliceosome. Here we show that Prp38p is a unique component of the U4/U6.U5 tri-small nuclear ribonucleoprotein (snRNP) particle and is necessary for an essential step late in spliceosome maturation. Without Prp38p activity spliceosomes form, but arrest in a catalytically impaired state. Functional spliceosomes shed U4 snRNA before 5' splice-site cleavage. In contrast, Prp38p-defective spliceosomes retain U4 snRNA bound to its U6 snRNA base-pairing partner. Prp38p is the first tri-snRNP-specific protein shown to be dispensable for assembly, but required for conformational changes which lead to catalytic activation of the spliceosome.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.     

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.     

Alterations of RNase H sensitivity of the 3'' splice site region during the in vitro splicing reaction.

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.     

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.     

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.     

Functional association of essential splicing factor(s) with PRP19 in a protein complex.

We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.     

Spliceostatin A inhibits spliceosome assembly subsequent to prespliceosome formation

Pre-mRNA splicing is catalyzed by the large ribonucleoprotein spliceosome. Spliceosome assembly is a highly dynamic process in which the complex transitions through a number of intermediates. Recently, the potent anti-tumor compound Spliceostatin A (SSA) was shown to inhibit splicing and to interact with an essential component of the spliceosome, SF3b. However, it was unclear whether SSA directly impacts the spliceosome and, if so, by what mechanism, which limits interpretation of the drugs influence on splicing. Here, we report that SSA inhibits pre-mRNA splicing by interfering with the spliceosome subsequent to U2 snRNP addition. We demonstrate that SSA inhibition of spliceosome assembly requires ATP, key pre-mRNA splicing sequences and intact U1 and U2 snRNAs. Furthermore all five U snRNAs in addition to the SSA molecule associate with pre-mRNA during SSA inhibition. Kinetic analyses reveal that SSA impedes the A to B complex transition. Remarkably, our data imply that, in addition to its established function in early U2 snRNP recruitment, SF3b plays a role in later maturation of spliceosomes. This work establishes SSA as a powerful tool for dissecting the dynamics of spliceosomes in cells. In addition our data will inform the design of synthetic splicing modulator compounds for targeted anti-tumor treatment.     

A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.     

The spliceosomal PRP19 complex of trypanosomes

In trypanosomes, mRNAs are processed by spliced leader (SL) trans splicing, in which a capped SL, derived from SL RNA, is spliced onto the 5′ end of each mRNA. This process is mediated by the spliceosome, a large and dynamic RNA‐protein machinery consisting of small nuclear ribonucleoproteins (snRNPs) and non‐snRNP proteins. Due to early evolutionary divergence, the amino acid sequences of trypanosome splicing factors exhibit limited similarity to those of their eukaryotic orthologs making their bioinformatic identification challenging. Most of the ～ 60 protein components that have been characterized thus far are snRNP proteins because, in contrast to individual snRNPs, purification of intact spliceosomes has not been achieved yet. Here, we characterize the non‐snRNP PRP19 complex of Trypanosoma brucei. We identified a complex that contained the core subunits PRP19, CDC5, PRL1, and SPF27, as well as PRP17, SKIP and PPIL1. Three of these proteins were newly annotated. The PRP19 complex was associated primarily with the activated spliceosome and, accordingly, SPF27 silencing blocked the first splicing step. Interestingly, SPF27 silencing caused an accumulation of SL RNA with a hypomethylated cap that closely resembled the defect observed previously upon depletion of the cyclin‐dependent kinase CRK9, indicating that both proteins may function in spliceosome activation.     

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.     

PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.     

Link of NTR-Mediated Spliceosome Disassembly with DEAH-Box ATPases Prp2, Prp16, and Prp22

The DEAH-box ATPase Prp43 is required for disassembly of the spliceosome after the completion of splicing or after the discard of the spliceosome due to a splicing defect. Prp43 associates with Ntr1 and Ntr2 to form the NTR complex and is recruited to the spliceosome via the interaction of Ntr2 and U5 component Brr2. Ntr2 alone can bind to U5 and to the spliceosome. To understand how NTR might mediate the disassembly of spliceosome intermediates, we arrested the spliceosome at various stages of the assembly pathway and assessed its susceptibility to disassembly. We found that NTR could catalyze the disassembly of affinity-purified spliceosomes arrested specifically after the ATP-dependent action of DEAH-box ATPase Prp2, Prp16, or Prp22 but not at steps before the action of these ATPases or upon their binding to the spliceosome. These results link spliceosome disassembly to the functioning of splicing ATPases. Analysis of the binding of Ntr2 to each splicing complex has revealed that the presence of Prp16 and Slu7, which also interact with Brr2, has a negative impact on Ntr2 binding. Our study provides insights into the mechanism by which NTR can be recruited to the spliceosome to mediate the disassembly of spliceosome intermediates when the spliceosome pathway is retarded, while disassembly is prevented in normal reactions.     

The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.     

U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.