The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.Splicing of nuclear precursor mRNA (pre-mRNA) is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five snRNAs, U1, U2, U4, U5, and U6, in the form of snRNPs and numerous protein factors (2,4,17,23,36,44,47). The spliceosome is assembled by ordered interactions of snRNPs with the pre-mRNA at the 5Ј splice site, the branch site, and the 3Ј splice site and also with each other. After binding of the five snRNAs, a large conformational rearrangement occurs that involves the dissociation of U1 and U4 and the formation of new base pairing between U2 and U6 and between U6 and the 5Ј splice site. Such structural rearrangement leads to the activation of the spliceosome, allowing catalytic reactions to proceed (2).The DEXD/H-box RNA helicase Prp2 is required for the first catalytic reaction (26,29). Prp2 functions as a molecular motor to restructure the spliceosome by hydrolyzing ATP, and then it leaves the spliceosome (25, 26). Another splicing factor, Spp2, originally identified as a high-copy-number suppressor of prp2-1 mutation, interacts with Prp2 and is required for the function of Prp2, possibly by mediating the binding of Prp2 to the spliceosome (28,32,35). After Prp2-dependent conformational rearrangement of the spliceosome, a heat-resistant heparin binding protein factor(s) of unknown identity, HP, is required to promote the step one reaction in an ATP-independent manner (26). Similarly, five protein factors have been shown to be involved in the second catalytic reaction. Hydrolysis of ATP by the DEXD/H-box RNA helicase Prp16 is required for the structural rearrangement of the spliceosome prior to the catalytic reaction (1,19,34). Prp17 has also been shown to act in the ATP-dependent step (22). The subsequent catalytic reaction ...

bThe 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. Introns are removed from precursor mRNAs (pre-mRNAs) via two transesterification reactions. The reactions take place on a large ribonucleoprotein complex called the spliceosome, which is composed of five small nuclear RNAs (snRNAs) and numerous protein factors. These factors bind to the pre-mRNA in a sequential manner to assemble the spliceosome into a functional complex for catalysis (for reviews, see references 1 to 4). After completion of the splicing reaction, the mature message is released and the spliceosome is disassembled to recycle its components.Extensive structural rearrangement of the spliceosome, including exchange of RNA base-pairing and protein components (1, 2, 4-9), is associated with each step of the spliceosome assembly process. DEXD/H-box RNA helicases have been proposed to mediate structural changes of the spliceosome in distinct steps (10-15). In the budding yeast Saccharomyces cerevisiae, eight DEXD/ H-box proteins are required for splicing. Prp5 and Sub2 are involved in early steps of spliceosome assembly to facilitate the formation of the prespliceosome (16-18). Prp28 and Brr2 are required in releasing U1 and U4, respectively, for the activation of the spliceosome (11, 15). Prp2 and Prp16 are required for the catalytic steps, and their activities are associated with the release of U2 components SF3a and SF3b (SF3a/b) and step-one factors Yju2 and Cwc25, respectively (19-24). After the completion of splicing, Prp22 is required for the release of mature mRNA and Prp43 for the disassembly of the spliceosome to recycle spliceosomal components (25-28). Although some of these proteins have been shown to unwind the RNA duplex in vitro, none show substrate specificity. Never...

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system.

The Prp19-associated complex (NineTeen Complex [NTC]) is required for spliceosome activation by specifying interactions of U5 and U6 with pre-mRNA on the spliceosome after the release of U4. The NTC consists of at least eight protein components, including two tetratricopeptide repeat (TPR)-containing proteins, Ntc90 and Ntc77. Ntc90 has nine copies of the TPR with seven clustered in the carboxy-terminal half of the protein, and interacts with all identified NTC components except for Prp19 and Ntc25. It forms a stable complex with Ntc31, Ntc30, and Ntc20 in the absence of Ntc25, when other interactions between NTC components are disrupted. In this study, we used both biochemical and genetic methods to analyze the structure of Ntc90, and its function in maintaining the integrity of the NTC and in NTC-mediated spliceosome activation. Our results show that Ntc90 interacts with Ntc31, Ntc30, and other NTC components through different regions of the protein, and that its function may be regulated by Ntc31 and Ntc30. Ntc90 is not required for the association of Prp19, Ntc85, Ntc77, Ntc25, and Ntc20, or for their binding to the spliceosome. It is also not required for NTC-mediated spliceosome activation, but is required for the recruitment of Yju2, which is involved in the first catalytic reaction after the function of Prp2. Our results demonstrate a novel role of the NTC in recruiting splicing factors to the spliceosome after its activation.

Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions. Cwc25 binds tightly to the spliceosome after the reaction and is then removed from the spliceosome, which normally requires DExD/H-box protein Prp16 and ATP hydrolysis, to allow the occurrence of the second reaction. When deprived of Cwc25, the purified first-step spliceosome catalyzes both forward and reverse splicing reactions under normal splicing conditions without requiring energy. Both reactions are inhibited when Cwc25 is added back, presumably due to the stabilization of first-step conformation. Prp16 is dispensable for the second reaction when splicing is carried out under conditions that destabilize Cwc25. We also show that the purified precatalytic spliceosome can catalyze two steps of the reaction at a low efficiency without requiring Cwc25, Slu7, or Prp18 when incubated under proper conditions. Our study reveals conformational modulation of the spliceosome by Cwc25 and Prp16 in stabilization and destabilization of first-step conformation, respectively, to facilitate the splicing process.