The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase.

Unlike autocatalyzed self-splicing reactions, nuclear pre-mRNA splicing requires transacting macromolecules and ATP. A protein encoded by the PRP2 gene of Saccharomyces cerevisiae is required, in conjunction with ATP, for the first cleavage-ligation reaction of pre-mRNA splicing. In this study, we have purified two forms of the PRP2 gene product with apparent molecular weights of 100 kDa and 92 kDa, from a yeast strain overproducing the protein. Both proteins were indistinguishable in their ability to complement extracts derived from a heat-sensitive prp2 mutant. Furthermore, we show that the PRP2 protein is capable of hydrolyzing nucleoside triphosphates in the presence of single-stranded RNAs such as poly(U). However, purified PRP2 by itself did not unwind double-stranded RNA substrates. The fact that an RNA-dependent NTPase activity is intrinsic to PRP2 may account for the ATP requirement in the first catalytic reaction of pre-mRNA splicing.     

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 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.     

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.     

Sequence requirements for splicing by the Cne PRP8 intein

The dependence of protein splicing on conserved residues of the Cne PRP8 intein was assessed by alanine scanning mutagenesis in a foreign protein context. Corroboration was obtained for the involvement of residues at the splice junctions and of the conserved threonine and histidine of motif B. Five additional residues were identified as absolutely required for splicing. Variant W151A displayed premature C-terminal cleavage, not seen with other Cne PRP8 mutants. We propose a model whereby W151 acts to prevent premature C-terminal cleavage, favoring complete splicing as opposed to two disjointed cleavage events.     

Formation of the yeast splicing complex A1 and association of the splicing factor PRP19 with the pre-mRNA are independent of the 3' region of the intron.

Assembly of the spliceosome is a step-wise process and involves sequential binding of snRNAs to the pre-mRNA to form pre-splicing complex A2-1. Subsequent dissociation of U4 from the spliceosome is accompanied by formation of complex A1 (Genes Dev. 1, 1014-1027, 1987). We show that the 3' region of the intron sequence is not required for efficient assembly of the yeast spliceosome. Truncated precursor mRNA retaining only four or five nucleotides 3' to the TACTAAC box formed pre-splicing complex A1, kinetically the last pre-mRNA containing splicing complex identified. The subsequent cleavage--ligation reaction requires at least 23 nucleotides on the 3' side of the TACTAAC box in a sequence-independent manner. Immunoprecipitation with anti-PRP19 antibody showed that association of PRP19 with the spliceosome was also independent of the 3' region of the intron.     

PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels.

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.     

A short 5' splice site RNA oligo can participate in both steps of splicing in mammalian extracts.

A short 5' splice site RNA oligonucleotide (5'SS RNA oligo) undergoes both steps of splicing when a second RNA containing the 3' splice site region (3'SS RNA) is added in trans. This trans-splicing reaction displays the same 5' and 3' splice site sequence requirements as cis-splicing of full-length pre-mRNA. The analysis of RNA-snRNP complexes formed on each of the two splice site RNAs is consistent with the formation of partial complexes, which then associate to form the complete spliceosome. Specifically, U2 snRNP bound to the 3'SS RNA associates with U4/U5/U6 snRNP bound to the 5'SS RNA oligo. Thus, as expected, trans-splicing depends on the integrity of U2, U4, and U6 snRNAs. However, unlike cis-splicing, trans-splicing is enhanced when the 5' end of U1 snRNA is blocked or removed or when the U1 snRNP is depleted. Thus, the early regulatory requirement for U1 snRNP, which is essential in cis-splicing, is bypassed in this trans-splicing system. This simplified trans-splicing reaction offers a unique model system in which to study the mechanistic details of pre-mRNA splicing.     

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.     

Interaction of the yeast DExH-box RNA helicase prp22p with the 3' splice site during the second step of nuclear pre-mRNA splicing

Using site-specific incorporation of the photochemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3′ splice sites. One of these is an unidentified ~122 kDa protein that cross-links to 3′ splice sites during formation of the pre-spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3′ splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3′ splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3′ splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3′ splice site of yeast introns is contacted much earlier than previously envisioned by a protein of ~120 kDa, and that a transient association of Prp22p with the 3′ splice site occurs between the first and second catalytic steps.     

A dominant negative mutation in the conserved RNA helicase motif ''SAT'' causes splicing factor PRP2 to stall in spliceosomes.

To characterize sequences in the RNA helicase-like PRP2 protein of Saccharomyces cerevisiae that are essential for its function in pre-mRNA splicing, a pool of random PRP2 mutants was generated. A dominant negative allele was isolated which, when overexpressed in a wild-type yeast strain, inhibited cell growth by causing a defect in pre-mRNA splicing. This defect was partially alleviated by simultaneous co-overexpression of wild-type PRP2. The dominant negative PRP2 protein inhibited splicing in vitro and caused the accumulation of stalled splicing complexes. Immunoprecipitation with anti-PRP2 antibodies confirmed that dominant negative PRP2 protein competed with its wild-type counterpart for interaction with spliceosomes, with which the mutant protein remained associated. The PRP2-dn1 mutation led to a single amino acid change within the conserved SAT motif that in the prototype helicase eIF-4A is required for RNA unwinding. Purified dominant negative PRP2 protein had approximately 40% of the wild-type level of RNA-stimulated ATPase activity. As ATPase activity was reduced only slightly, but splicing activity was abolished, we propose that the dominant negative phenotype is due primarily to a defect in the putative RNA helicase activity of PRP2 protein.     

Protein splicing of PRP8 mini-inteins from species of the genus Penicillium

Inteins are protein-intervening sequences found inside the coding region of different host proteins and are translated in-frame with them. They can self-excise through protein splicing, which ligates the host protein flanks with a peptide bond. In this study, four different species of the genus Penicillium were investigated for the presence of inteins inside the conserved splicing-factor protein PRP8. We identified 157 to 162 amino acid in-frame insertions in the PRP8 protein of Penicillium chrysogenum, Penicillium expansum, and Penicillium vulpinum (formerly Penicillium claviforme). The Penicillium PRP8 inteins are mini-inteins without a conserved endonuclease domain. We demonstrated that the PRP8 mini-inteins of P. chrysogenum, P. expansum, and P. vulpinum undergo autocatalytic protein splicing when heterologously expressed in E. coli, in a model host protein, and in a divided GFP model system. They are, thus, among the smallest known nuclear-encoded, active splicing protein elements. The GFP assay should be valuable as a screening system for protein splicing inhibitors as potential antimycotic agents and as tools for studying the mechanism of protein splicing of fungal mini-inteins.     

Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution.

The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.     

A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing.

Yeast fibrillarin (NOP1) is an evolutionarily conserved, nucleolar protein necessary for multiple steps in ribosome biogenesis. Yeast mutants lacking a functional NOP1 gene can be complemented by human fibrillarin but are temperature sensitive for growth and impaired in pre-rRNA processing. In order to identify components which interact functionally with human fibrillarin in yeast, we isolated extragenic suppressors of this phenotype. One dominant suppressor, sof1-56, which is allele-specific for human fibrillarin and restores growth and pre-RNA processing at 35 degrees C, was cloned by in vivo complementation. The wild-type allele of SOF1 is essential for cell growth and encodes a novel 56 kDa protein. In its central domain, SOF1 contains a repeated sequence also found in beta-subunits of trimeric G-proteins and the splicing factor PRP4. A single amino acid exchange in the G beta-like repeat domain is responsible for the suppressing activity of sof1-56. Indirect immunofluorescence shows that SOF1 is located within the yeast nucleolus. Co-immunoprecipitation demonstrates the physical association of SOF1 with U3 small nucleolar RNA and NOP1. In vivo depletion of SOF1 leads to impaired pre-rRNA processing and inhibition of 18S rRNA production. Thus, SOF1 is a new component of the nucleolar rRNA processing machinery.     

SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing.

Understanding the mechanism of pre-mRNA splicing requires the characterization of all components involved. In the present study, we used the genetically and biochemically defined yeast PRP16 protein as a point of departure for the identification of additional factors required for the second catalytic step in vitro. We isolated by glycerol gradient sedimentation spliceosomes that were formed in yeast extracts depleted of PRP16. This procedure separated the spliceosomal complexes containing lariat intermediate and exon 1 from free proteins present in the whole-cell yeast extract. We then supplemented these spliceosomes with purified proteins or yeast extract fractions as a functional assay for second-step splicing factors. We show that SLU7 protein and a novel activity that we named SSF1 (second-step factor 1) were required in concert with PRP16 to promote progression through the second catalytic step of splicing. Taking advantage of a differential ATP requirement for PRP16 and SLU7 function, we show that SLU7 can act after PRP16 in the splicing pathway.     

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.     

Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation

The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.