Two novel yeast splicing factors required for spliceosome disassembly have been identified. Ntr1 and Ntr2 (NineTeen complex-Related proteins) were identified for their weak association with components of the Prp19-associated complex. Unlike other Prp19-associated components, these two proteins were primarily associated with the intron-containing spliceosome during the splicing reaction. Extracts depleted of Ntr1 or Ntr2 exhibited full splicing activity, but accumulated large amounts of lariat-intron in the spliceosome after splicing, indicating that the normal function of the Prp19-associated complex in spliceosome activation was not affected, but spliceosome disassembly was hindered. Immunoprecipitation analysis revealed that Ntr1 and Ntr2 formed a stable complex with DExD/H-box RNA helicase Prp43 in the splicing extract. Ntr1 interacted with Prp43 through the N-terminal G-patch domain, with Ntr2 through a middle region, and with itself through the carboxyl half of the protein. The affinity-purified Ntr1-Ntr2-Prp43 complex could catalyze disassembly of the spliceosome in an ATP-dependent manner, separating U2, U5, U6, NTC (NineTeen Complex), and lariat-intron. This is the first demonstration of physical disassembly of the spliceosome, catalyzed by a complex containing a DExD/H-box RNA helicase and two accessory factors, which might function in targeting the helicase to the correct substrate.[Keywords: Spliceosome disassembly; NTR complex; Prp43; Ntr1; Ntr2] Supplemental material is available at http://www.genesdev.org.

The non-coding RNA subunit of telomerase provides the template for telomerase activity. In diverse fungi, 3' end processing of telomerase RNA involves a single cleavage by the spliceosome. Here, we examine how human telomerase RNA (hTR) primary transcripts are processed into the mature form of precisely 451 nt. We find that the splicing inhibitor isoginkgetin mimics the effects of RNA exosome inhibition and causes accumulation of long hTR transcripts. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly(A)-specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and degradation and suggest treatment options for telomerase insufficiency disorders.

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.

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions. The presence of introns in spliceosomal RNAs of certain fungi has suggested that splicing may be reversible; however, this has never been demonstrated experimentally. By using affinity-purified spliceosomes, we have shown that both catalytic steps of splicing can be efficiently reversed under appropriate conditions. These results provide considerable insight into the catalytic flexibility of the spliceosome.

Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn 2؉ . These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5 splice site and the branch point during the first catalytic reaction.Precursor mRNAs (pre-mRNAs) excise their introns via two steps of a transesterification reaction. The reaction takes place on a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, U6, and numerous protein factors. The spliceosome is a highly dynamic structure, formed by stepwise binding to the pre-mRNA of snRNAs in the form of small nuclear ribonucleoprotein complexes (snRNPs) (for a review, see references 3, 29, and 35-37). Following the binding of all snRNAs, the spliceosome undergoes a major structural change, leading to the release of U1 and U4 and the formation of the active spliceosome that is able to carry out the catalytic reaction.Spliceosome activation also requires a large protein complex, the Prp19-associated complex (nineteen complex, or NTC), which is added to the spliceosome after the release of U1 and U4 to stabilize the association of U5 and U6 with the spliceosome (5). The NTC plays an important role in promoting or stabilizing high-specificity interactions between U6 and the 5Ј splice site and between U5 and the exon sequence at the splice junctions after U1 and U4 have dissociated (4, 5). Eight components of the NTC have been identified, including Prp19,

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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.Splicing of nuclear pre-mRNA requires five small nuclear RNAs (snRNAs) and numerous protein factors (for reviews, see references 3 and 4). These factors bind to the pre-mRNA in a sequential manner, in the order of U1, U2, and then U4/U6.U5 as a tri-snRNP particle, to assemble the spliceosome. Subsequent to the binding of tri-snRNP, the spliceosome undergoes a major structural rearrangement, including the release of U1 and U4, and the addition of a large protein complex, the Prp19-associated complex (or NTC), and becomes catalytically competent. After the splicing reaction is complete, the postcatalytic spliceosome first releases the mature mRNA and then undergoes disassembly to dissociate all components for a new round of splicing. Extensive structural rearrangement of the pre-mRNA, including the formation and/or disruption of RNA base pairing, is associated with each step of the assembly and disassembly process. Mechanical devices that promote base pairing or facilitate unwinding assist in mediating such RNA rearrangements (for a review, see reference 32), and it is proposed that the U5 component Snu114 serves as a signal-dependent switch to control the spliceosome dynamics (31).The DEXD/H-box RNA helicases belong to a large superfamily of proteins conserved from bacteria and viruses to humans (6, 35). They share a highly conserved helicase domain that includes the motif DEXD/H and play roles in all biological processes of RNA molecules, including transcription, editing, splicing, ribosome biogenesis, RNA export, translation, and RNA turnover. All of these proteins have ATPase or NTPase activities stimulated by RNA. Eight DEXD/H-box proteins are involved in various steps of the splicing reaction (29, 32). Each of them was thought to facilitate a structural transition at distinct steps, coupling the energy from ATP hydrolysis to remodeling of RNA-RNA or RNA-protein interactions. Among these proteins, Prp5, Prp2...

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...