Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2

Removal of intron regions from pre-messenger RNA (pre-mRNA) requires spliceosome assembly with pre-mRNA, then subsequent spliceosome remodeling to allow activation for the two steps of intron removal. Spliceosome remodeling is carried out through the action of DExD/H-box ATPases that modulate RNA–RNA and protein–RNA interactions. The ATPase Prp16 remodels the spliceosome between the first and second steps of splicing by catalyzing release of first step factors Yju2 and Cwc25 as well as destabilizing U2-U6 snRNA helix I. How Prp16 destabilizes U2-U6 helix I is not clear. We show that the NineTeen Complex (NTC) protein Cwc2 displays genetic interactions with the U6 ACAGAGA, the U6 internal stem loop (ISL) and the U2-U6 helix I, all RNA elements that form the spliceosome active site. We find that one function of Cwc2 is to stabilize U2-U6 snRNA helix I during splicing. Cwc2 also functionally cooperates with the NTC protein Isy1/NTC30. Mutation in Cwc2 can suppress the cold sensitive phenotype of the prp16-302 mutation indicating a functional link between Cwc2 and Prp16. Specifically the prp16-302 mutation in Prp16 stabilizes Cwc2 interactions with U6 snRNA and destabilizes Cwc2 interactions with pre-mRNA, indicating antagonistic functions of Cwc2 and Prp16. We propose that Cwc2 is a target for Prp16-mediated spliceosome remodeling during pre-mRNA splicing.     

Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway

Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction. The stable association of Cwc22 with the spliceosome requires the presence of the NTC but is independent of Prp2. Although Cwc22 is not required for the recruitment of Prp2 to the spliceosome, it is essential for the function of Prp2 in promoting the release of the U2 components SF3a and SF3b. In the absence of Cwc22, Prp2 can bind to the spliceosome but is dissociated upon ATP hydrolysis without promoting the release of SF3a/b. Thus, Cwc22 represents a novel ATP-dependent step one factor besides Prp2 and Spp2 and has a distinct role from that of Spp2 in mediating the function of Prp2.     

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.     

PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.

Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein.     

Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system

Step 2 catalysis of pre-mRNA splicing entails the excision of the intron and ligation of the 5′ and 3′ exons. The tasks of the splicing factors Prp16, Slu7, Prp18, and Prp22 in the formation of the step 2 active site of the spliceosome and in exon ligation, and the timing of their recruitment, remain poorly understood. Using a purified yeast in vitro splicing system, we show that only the DEAH-box ATPase Prp16 is required for formation of a functional step 2 active site and for exon ligation. Efficient docking of the 3′ splice site (3′SS) to the active site requires only Slu7/Prp18 but not Prp22. Spliceosome remodeling by Prp16 appears to be subtle as only the step 1 factor Cwc25 is dissociated prior to step 2 catalysis, with its release dependent on docking of the 3′SS to the active site and Prp16 action. We show by fluorescence cross-correlation spectroscopy that Slu7/Prp18 and Prp16 bind early to distinct, low-affinity binding sites on the step-1-activated B* spliceosome, which are subsequently converted into high-affinity sites. Our results shed new light on the factor requirements for step 2 catalysis and the dynamics of step 1 and 2 factors during the catalytic steps of splicing.     

Physical and genetic interactions of yeast Cwc21p,an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors—namely, Prp8p and Snu114p—and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.     

Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.

In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.     

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.     

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.     

Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing

Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.     

Dynamic Contacts of U2, RES,Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome''s protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.     

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 assembly of the SF3a splicing factor complex of U2 snRNP

SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structure-guided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.     

Interaction between a G-patch protein and a spliceosomal DEXD/H-box ATPase that is critical for splicing

Prp2 is an RNA-dependent ATPase that activates the spliceosome before the first transesterification reaction of pre-mRNA splicing. Prp2 has extensive homology throughout the helicase domain characteristic of DEXD/H-box helicases and a conserved carboxyl-terminal domain also found in the spliceosomal helicases Prp16, Prp22, and Prp43. Despite the extensive homology shared by these helicases, each has a distinct, sequential role in splicing; thus, uncovering the determinants of specificity becomes crucial to the understanding of Prp2 and the other DEAH-splicing helicases. Mutations in an 11-mer near the C-terminal end of Prp2 eliminate its spliceosome binding and splicing activity. Here we show that a helicase-associated protein interacts with this domain and that this interaction contributes to the splicing process. First, a genome-wide yeast two-hybrid screen using Prp2 as bait identified Spp2, which contained a motif with glycine residues found in a number of RNA binding proteins. SPP2 was originally isolated as a genetic suppressor of a prp2 mutant. In a reciprocal screen, Spp2 specifically pulled out the C-terminal half of Prp2. Mutations in the Prp2 C-terminal 11-mer that disrupted function or spliceosome binding also disrupted Spp2 interaction. A screen of randomly mutagenized SPP2 clones identified an Spp2 protein with a mutation in the G patch that could restore interaction with Prp2 and enhanced splicing in a prp2 mutant strain. The study identifies a potential mechanism for Prp2 specificity mediated through a unique interaction with Spp2 and elucidates a role for a helicase-associated protein in the binding of a DEXD/H-box protein to the spliceosome.     

A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome.

PRP16 is an RNA-dependent ATPase required for the second catalytic step of splicing in vitro. A dominant suppressor of a branchpoint mutation in Saccharomyces cerevisiae, the prp16-1 allele, contains a Tyr to Asp change in the nucleotide-binding site consensus sequence. We now find that cells harboring the prp16-1 allele have a general growth defect that is exacerbated at cold temperatures. The mutant is dominant over the wild-type gene when overexpressed. Purified Prp16-1 protein binds to the spliceosome with apparently wild-type affinity; however, it only weakly complements the second-step block in a PRP16-depleted extract. Analysis of purified Prp16-1 revealed that the rate of ATP hydrolysis is greatly reduced. These results can account for the dominant negative growth phenotype and argue that the ATPase activity of PRP16 is essential for its role in splicing. Moreover, since PRP16 is a member of the DEAD/H box families, these findings have important implications for a large class of proteins.     

Brr2解旋U4/U6 SnRNAs的调控机制

前体mRNA(precursor messager RNA,pre-mRNA)剪接是去除内含子和将外显子彼此连接形成成熟mRNA的过程。剪接过程在一个呈动态变化的大核糖核蛋白(ribonucleoprotein, RNP)复合体,即剪接体催化作用下完成。DExD/H-box RNA解旋酶在剪接体组装、激活及解聚过程中都发挥着重要作用。Brr2(bad response to refrigeration 2)这种DExD/H-box RNA解旋酶是构成U5稳定的亚单位。Brr2含有两个串联解旋酶盒结构,在剪接体激活中负责U4/U6的解旋,还参与剪接体催化及解聚过程,因此Brr2在剪接过程中必需具备严格的调控机制。在剪接过程中,Prp8的C端包含两个连续的RNase H域和Jab1/MPN域,能够正负调控Brr2活性。Snu114在调节Brr2活性中具有非常重要的作用。此外,Brr2通过C端解旋酶盒(C-terminal cassette, CC)与N末端域(N-terminal region)进行分子内的自我活性调节。本文综述了近年来在Brr2的分子间和分子内活性调节机制的研究进展,这些不同的调节机制协同作用才确保真核生物pre-mRNA可变剪接的保真性。     

Genetic analysis reveals a role for the C terminus of the Saccharomyces cerevisiae GTPase Snu114 during spliceosome activation

Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III-V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10- to 20-amino-acid stretches in each of domains III-V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis.     

Functional roles of DExD/H-box RNA helicases in Pre-mRNA splicing

Splicing of precursor mRNA takes place via two consecutive steps of transesterification catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome is assembled through ordered binding to the pre-mRNA of five small nuclear RNAs and numerous protein factors, and is disassembled after completion of the reaction to recycle all components. Throughout the splicing cycle, the spliceosome changes its structure, rearranging RNA-RNA, RNA-protein and protein-protein interactions, for positioning and repositioning of splice sites. DExD/H-box RNA helicases play important roles in mediating structural changes of the spliceosome by unwinding of RNA duplexes or disrupting RNA-protein interactions. DExD/H-box proteins are also implicated in the fidelity control of the splicing process at various steps. This review summarizes the functional roles of DExD/H-box proteins in pre-mRNA splicing according to studies conducted mostly in yeast and will discuss the concept of the complicated splicing reaction based on recent findings.     

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.