A spontaneous duplication in U6 spliceosomal RNA uncouples the early and late functions of the ACAGA element in vivo.

U6 RNA enters the spliceosome base paired with U4 RNA, but dissociates from U4 RNA before the catalytic steps of splicing. We have identified a cold-sensitive lethal mutation in U4 RNA (U4-cs1) that blocks the splicing pathway after U4/U6 complex formation, but before the first catalytic step of splicing. Remarkably, selection for suppressors of the cold-sensitive growth of the U4-cs1 strain yielded a tandem duplication of the highly conserved ACAGA sequence of U6 RNA (U6-Dup). The ACAGA sequence plays an essential role in spliceosome assembly and in the second catalytic step of pre-mRNA splicing; one or both of these roles involves direct base pairing to the pre-mRNA 5' splice site. In a U4-cs1/U6-Dup double-mutant strain grown at low temperature, the upstream ACAGA sequence of U6 RNA is required for suppression of the U4 mutation, whereas the downstream ACAGA sequence is required for other essential functions. Based on the sequence requirements for function of the upstream ACAGA element of U6-Dup, we propose that it pairs with the pre-mRNA 5' splice site during incorporation of the U4/U6 complex into the spliceosome and that the subsequent dissociation of U4 RNA exposes the downstream ACAGA sequence, which functions in the catalytic steps. The properties of this mutant U4/U6 complex provide compelling in vivo evidence that U6 RNA normally base pairs with the 5' splice site before disruption of its pairing with U4 RNA.     

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.     

An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p

Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.     

Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor

While some members of the ubiquitous DExD/H box family of proteins have RNA helicase activity in vitro, their roles in vivo remain virtually unknown. Here, we show that the function of an otherwise essential DEAD box protein, Prp28p, can be bypassed by mutations that alter either the protein U1-C or the U1 small nuclear RNA. Further analysis suggests that the conserved L13 residue in the U1-C protein makes specific contact to stabilize the U1 snRNA/5' splice site duplex in the prespliceosome, and that Prp28p functions to counteract the stabilizing effect of the U1-C protein, thereby promoting the dissociation of the U1 small nuclear ribonucleoprotein particle from the 5' splice site. Thus, in addition to unwinding RNA, the DExD/H box proteins may affect RNA-RNA rearrangements by antagonizing specific RNA-stabilizing proteins.     

Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements.

U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.     

A novel U1/U5 interaction indicates proximity between U1 and U5 snRNAs during an early step of mRNA splicing.

The five spliceosomal snRNAs (U1, U2, U4, U5, and U6) undergo an ordered sequence of conformational changes as mRNA splicing progresses. We have shown that an antisense RNA oligonucleotide complementary to U5 snRNA induces a novel U1/U4/U5 complex that may be a transitional stage in the displacement of U1 from the 5' splice site by U5. Here we identify a novel site-specific crosslink between the 5' end of U1 and the invariant loop of U5 snRNA. This crosslink can be induced in nuclear extract by an antisense oligonucleotide directed against U5 snRNA, but can also be detected during an early step of the splicing reaction in the absence of oligonucleotide. Our data indicate proximity between U1 and U5 snRNPs before the first catalytic step of splicing, and may suggest that U1 helps to direct U5 to the 5' splice site.     

Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.     

Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing.

For the second catalytic step of pre-mRNA splicing to occur, a 3' splice site must be selected and juxtaposed with the 5' exon. Four proteins, Prp16p, Slu7p, Prp17p, Prp18p, and an integral spliceosomal protein, Prp8p, are known to be required for the second catalytic step. prp8-101, an allele of PRP8 defective in 3' splice site recognition, exhibits specific genetic interactions with mutant alleles of the other second step splicing factors. The prp8-101 mutation also results in decreased crosslinking of Prp8p to the 3' splice site. To determine the role of the step-two-specific proteins in 3' splice site recognition and in binding of Prp8p to the 3' splice site, we performed crosslinking studies in mutant and immunodepleted extracts. Our results suggest an ordered pathway in which, after the first catalytic step, Prp16p crosslinks strongly to the 3' splice site and Prp8p and Slu7p crosslink weakly. ATP hydrolysis by Prp16p affects a conformational change that reduces the crosslinking of Prp16p with the 3' splice site and allows stronger crosslinking of Prp8p and Slu7p. Thus, the 3' splice site appears to be recognized in two stages during the second step of splicing. Strong 3' splice site crosslinking of Prp8p and Slu7p also requires the functions of Prp17p and Prp18p. Therefore, Prp8p and Slu7p interact with the 3' splice site at the latest stage of splicing prior to the second catalytic step that can currently be defined, and may be at the active site.     

Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.     

The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex

Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.     

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.     

trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing

Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5' end of U2 snRNA and the 3' exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5' splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5' splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5' splice sites are predicted to bind poorly at the spliceosome catalytic center. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.     

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.     

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.     

The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex.

The yeast PRP44 gene, alternatively named as BRR2, SLT22, RSS1, or SNU246, encodes a 246-kDa protein with putative RNA helicase function during pre-mRNA splicing. The protein is a typical DEAD/H family member, but unlike most other members of this family, it contains two putative RNA helicase domains, each with a highly conserved ATPase motif. Prior to this study little was known about functional roles for these two domains. We present genetic and biochemical evidence that ATPase motifs of only the first helicase domain are required for cell viability and pre-mRNA splicing. Overexpression of mutations in the first domain results in a dominant negative phenotype, and extracts from these mutant strains inhibit in vitro pre-mRNA splicing. In vitro analyses of affinity purified proteins revealed that only the first helicase domain possesses poly (U)-dependent ATPase activity. Overexpression of a dominant negative protein in vivo reduces the relative abundance of free U4 and U6 snRNA with a concomitant accumulation of the U4/U6 duplex. Accumulation of the U4/U6 duplex was relieved by overexpression of wild-type Prp44p. Three DEAD/H box proteins, Prp16p, Prp22p and Prp44p, have previously been shown to affect U4/U6 unwinding activity in vitro. The possible role of these proteins in mediating this reaction in vivo was explored following induced expression of ATPase domain mutants in each of these. Although overexpression of the mutant form of either Prp16p, Prp22p, or Prp44p was lethal, only expression of the mutant Prp44p resulted in accumulation of the U4/U6 helix. Our results, when combined with previously published in vitro results, support a direct role for Prp44p in unwinding of the U4/U6 helix.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.     

Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome

Important general insights into the mechanism of pre-mRNA splicing have emerged from studies of the U12-dependent spliceosome. Here, photochemical cross-linking analyses during U12-dependent spliceosome assembly have surprisingly revealed that an upstream 5' exon region is required for establishing two essential catalytic core interactions, U12/U6atac helix Ib and U6atac/5' splice site contacts, but not for U5/5' exon interactions or partial unwinding of U4atac/U6atac. A novel intermediate, representing an alternative pathway for catalytic core formation, is a ternary snRNA complex containing U4atac/U6atac stem II and U12/U6atac helix Ia that forms even without U6atac replacing U11 at the 5' splice site. A powerful oligonucleotide displacement method suggests that the blocked complexes analyzed to deduce the interdependence of these multiple RNA exchanges are authentic intermediates in U12-dependent spliceosome assembly.