Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B(act) spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome's catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.     

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome

Current models of the core of the spliceosome include a network of RNA-RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8-RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome's RNA core.     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome

The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding.     

Prp21, a U2-snRNP-associated protein, and Prp24, a U6-snRNP-associated protein, functionally interact during spliceosome assembly in yeast

Earlier studies on genetic suppression ofprp24-1 byprp21-2 suggested an association between yeast Prp21 and Prp24 proteins, which are associated, respectively, with U2 snRNA and U6 snRNA. Here we report analyses of physical and functional interaction between these factors. Missense mutations in functionally important domains reside inprp21-2 andprp24-1. Two-hybrid assays do not detect interaction between wild-type or mutant proteins. Prp21-2 and Prp24-1 protein inprp21-2 orprp24-1 extracts can be heat-inactivatedin vitro. In contrast, heat-treated extracts from the revertant strainprp21-2 prp24-1 demonstrate allele-specific restoration of splicing. Suppression ofprp24-1 byprp21-2 does not cause coimmunoprecipitation of U2 and U6 snRNAs. We demonstrate the presence of Prp21 in the spliceosome assembly intermediate A2-1, and our data suggest the presence of Prp24 in the same complex. Kinetic analysis of assembly in heat-treated revertant extracts reveal a rate-limiting conversion of complex B to A2-1, suggesting transient association between the mutant proteins at this step. Our data also imply a requirement for Prp21 during B to A2-1 conversion. We conclude that a transient yet likely functional association between Prp21 and Prp24 occurs during spliceosome assembly.     

Definition of a spliceosome interaction domain in yeast Prp2 ATPase

The Saccharomyces cerevisiae splicing factor Prp2 is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. It contains three domains: a unique N-terminal domain, a helicase domain that is highly conserved in the DExD/H protein family, and a C-terminal domain that is conserved in spliceosomal DEAH proteins Prp2, Prp16, Prp22, and Prp43. We examined the role of each domain of Prp2 by deletion mutagenesis. Whereas deletions of either the helicase or C-terminal domain are lethal, deletions in the N-terminal domain have no detectable effect on Prp2 activity. Overexpression of the C-terminal domain of Prp2 exacerbates the temperature-sensitive phenotype of a prp2(Ts) strain, suggesting that the C-domain interferes with the activity of the Prp2(Ts) protein. A genetic approach was then taken to study interactions between Prp2 and the spliceosome. Previously, we isolated dominant negative mutants in the helicase domain of Prp2 that inhibit the activity of wild-type Prp2 when the mutant protein is overexpressed. We mutagenized one prp2 release mutant gene and screened for loss of dominant negative function. Several weak binding mutants were isolated and mapped to the C terminus of Prp2, further indicating the importance of the C terminus in spliceosome binding. This study is the first to indicate that amino acid substitutions outside the helicase domain can abolish spliceosome contact and splicing activity of a spliceosomal DEAH protein.     

ATP-dependent remodeling of the spliceosome: intragenic suppressors of release-defective mutants of Saccharomyces cerevisiae Prp22

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (635SAT637) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (635AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (804QAKGRAGR811). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.     

Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly

Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre‐mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre‐mRNA splicing, leading to growth‐deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA‐dependent ATPase involved in monitoring the U2 BSRR‐branch site base‐pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre‐mRNA splicing by directly altering the binding/ATPase activity of Prp5.     

A conformational rearrangement in the spliceosome sets the stage for Prp22-dependent mRNA release

An essential step in pre-mRNA splicing is the release of the mRNA product from the spliceosome. The DEAH box RNA helicase Prp22 catalyzes mRNA release by remodeling contacts within the spliceosome that involve the U5 snRNP. Spliceosome disassembly requires a segment of more than 13 ribonucleotides downstream of the 3' splice site. I show here by site-specific crosslinking and RNase H protection that Prp22 interacts with the mRNA downstream of the exon-exon junction prior to mRNA release. The findings support a model for Prp22-catalyzed mRNA release from the spliceosome wherein a rearrangement that accompanies the second transesterification step deposits Prp22 on the mRNA downstream of the exon-exon junction. Bound to its target RNA, the 3'-->5' helicase acts to disrupt mRNA/U5 snRNP contacts, thereby liberating the mRNA from the spliceosome.     

Prp8p dissection reveals domain structure and protein interaction sites

We describe a novel approach to characterize the functional domains of a protein in vivo. This involves the use of a custom-built Tn5-based transposon that causes the expression of a target gene as two contiguous polypeptides. When used as a genetic screen to dissect the budding yeast PRP8 gene, this showed that Prp8 protein could be dissected into three distinct pairs of functional polypeptides. Thus, four functional domains can be defined in the 2413-residue Prp8 protein, with boundaries in the regions of amino acids 394-443, 770, and 2170-2179. The central region of the protein was resistant to dissection by this approach, suggesting that it represents one large functional unit. The dissected constructs allowed investigation of factors that associate strongly with the N- or the C-terminal Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770, whereas fragmenting Prp8p at residue 2173 destabilizes its association with Aar2p.     

Mutations within the yeast U4/U6 snRNP protein Prp4 affect a late stage of spliceosome assembly.

We showed previously that the yeast Prp4 protein is a spliceosomal factor that is tightly associated with the U4, U5, and U6 small nuclear RNAs. Moreover, Prp4 appears to associate very transiently with the spliceosome before the U4 snRNA dissociates from the spliceosome. Prp4 belongs to the Gbeta-like protein family, which suggests that the Prp4 Gbeta motifs could mediate interactions with other components of the spliceosome. To investigate the function of the Gbeta motifs, we introduced mutations within the second WD-repeat of Prp4. Among the 35 new alleles found, 24 were pseudo wild-type mutants, 8 failed to grow at any temperature, and 3 were conditional sensitive mutants. The biochemical defects of the three thermosensitive prp4 mutants have been examined by immunoprecipitation, native gel electrophoresis, and glycerol gradient centrifugation. First, we show that snRNP formation is not impaired in these mutants and that Prp4 is present in the U4/U6 and U4/U6-U5 snRNP particles. We also demonstrate that spliceosome assembly is largely unaffected despite the fact that the first step of splicing does not occur. However, both Prp4 and U4 snRNA remain tightly associated with the spliceosome and this blocks the transition toward an active form of the spliceosome. Our results suggest a possible role of Prp4 in mediating important conformational rearrangements of proteins within the spliceosome that involve the region containing the Gbeta-repeats.     

DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family. The detailed mechanism of their action was not well understood until recently, when Prp2 was shown to be required for the release of U2 components SF3a and SF3b, presumably to allow the binding of Cwc25 to promote the first transesterification reaction. We show here that Cwc25 and Yju2 are released after the reaction in Prp16- and ATP-dependent manners, possibly to allow for the binding of Prp22, Prp18, and Slu7 to promote the second catalytic reaction. The binding of Cwc25 to the spliceosome is destabilized by mutations at the branchpoint sequence, suggesting that Cwc25 may bind to the branch site. We also show that Prp16 has an ATP-independent role in the first catalytic step, in addition to its known role in the second step. In the absence of ATP, Prp16 stabilizes the binding of Cwc25 to the spliceosome formed with branchpoint mutated pre-mRNAs to facilitate their splicing. Our results uncovered novel functions of Prp16 in both catalytic steps, and provide mechanistic insights into splicing catalysis.     

Prp43 is an essential RNA-dependent ATPase required for release of lariat-intron from the spliceosome

The essential Saccharomyces cerevisiae PRP43 gene encodes a 767-amino acid protein of the DEXH-box family. Prp43 has been implicated in spliceosome disassembly (Arenas, J. E., and Abelson, J. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11798-11802). Here we show that purified recombinant Prp43 is an RNA-dependent ATPase. Alanine mutations at conserved residues within motifs I ((119)GSGKT(123)), II ((215)DEAH(218)) and VI ((423)QRAGRAGR(430)) that diminished ATPase activity in vitro were lethal in vivo, indicating that ATP hydrolysis is necessary for the biological function of Prp43. Overexpression of lethal, ATPase-defective mutants in a wild-type strain resulted in dominant-negative growth inhibition. The ATPase-defective mutant T123A interfered in trans with the in vitro splicing function of wild-type Prp43. T123A did not affect the chemical steps of splicing or the release of mature mRNA from the spliceosome, but it blocked the release of the excised lariat-intron from the spliceosome. We show that the lariat-intron is not accessible to debranching by purified Dbr1 when it is held in the T123A-arrested splicing complex. Our results define a new ATP-dependent step of splicing that is catalyzed by Prp43.     

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.     

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.     

Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly

The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly. Binding of Ntr1-Ntr2 or Prp43 does not require ATP, but disassembly of the spliceosome requires hydrolysis of ATP. The NTR complex also dynamically interacts with U5 snRNP. Ntr2 interacts with U5 component Brr2 and is essential for both interactions of NTR with U5 and with the spliceosome. Ntr2 alone can also bind to U5 and to the spliceosome, suggesting a role of Ntr2 in mediating the binding of NTR to the spliceosome through its interaction with U5. Our results demonstrate that dynamic interactions of NTR with U5, through the interaction of Ntr2 with Brr2, and interactions of Ntr1 and Prp43 govern the recruitment of Prp43 to the spliceosome to mediate spliceosome disassembly.     

Structure and function of an RNase H domain at the heart of the spliceosome

Precursor-messenger RNA (pre-mRNA) splicing encompasses two sequential transesterification reactions in distinct active sites of the spliceosome that are transiently established by the interplay of small nuclear (sn) RNAs and spliceosomal proteins. Protein Prp8 is an active site component but the molecular mechanisms, by which it might facilitate splicing catalysis, are unknown. We have determined crystal structures of corresponding portions of yeast and human Prp8 that interact with functional regions of the pre-mRNA, revealing a phylogenetically conserved RNase H fold, augmented by Prp8-specific elements. Comparisons to RNase H-substrate complexes suggested how an RNA encompassing a 5'-splice site (SS) could bind relative to Prp8 residues, which on mutation, suppress splice defects in pre-mRNAs and snRNAs. A truncated RNase H-like active centre lies next to a known contact region of the 5'SS and directed mutagenesis confirmed that this centre is a functional hotspot. These data suggest that Prp8 employs an RNase H domain to help assemble and stabilize the spliceosomal catalytic core, coordinate the activities of other splicing factors and possibly participate in chemical catalysis of splicing.     

Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly

ATPase-facilitated steps during spliceosome function have been postulated to afford opportunities for kinetic proofreading. Spliceosome assembly requires the ATPase Prp5p, whose activity might thus impact fidelity during initial intron recognition. Using alanine mutations in S. cerevisiae Prp5p, we identified a suboptimal intron whose splicing could be improved by altered Prp5p activity and then, using this intron, screened for potent prp5 mutants. These prp5 alleles specifically alter branch region selectivity, with improved splicing in vivo of suboptimal substrates correlating with reduced ATPase activity in vitro for a series of mutants in ATPase motif III (SAT). Because these effects are abrogated by compensatory U2 snRNA mutations or other changes that increase branch region-U2 pairing, these results explicitly link a fidelity event with a defined physical structure, the branch region-U2 snRNA duplex, and provide strong evidence that progression of the splicing pathway requires branch region-U2 snRNA pairing prior to Prp5p-facilitated conformational change.