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,

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 ...

Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy. Here, we show that the zinc finger domain is essential for Cwc24 function, while the RING finger domain is dispensable. Cwc24 binds to the spliceosome after the Prp19-associated complex and is released upon Prp2 action. Cwc24 is not required for Prp2-mediated remodeling of the spliceosome, but the spliceosome becomes inactive if remodeling occurs before the addition of Cwc24. Cwc24 binds directly to pre-mRNA at the 5= splice site, spanning the splice junction. In the absence of Cwc24, U5 and U6 modes of interaction with the 5= splice site are altered, and splicing is very inefficient, with aberrant cleavage at the 5= splice site. Our data suggest roles for Cwc24 in orchestrating organization of the spliceosome into an active configuration prior to Prp2-mediated spliceosome remodeling and in promoting specific interaction of U5 and U6 with the 5= splice site for fidelity of 5= splice site selection. KEYWORDS Cwc24, Prp2, spliceosome, first catalytic step, splicing fidelity T he spliceosome is a large ribonucleoprotein complex that catalyzes the removal of intervening sequences (introns) from precursor mRNAs by two-step transesterification reactions. Consisting of five small nuclear RNAs (snRNAs) and a range of protein factors, the spliceosome recognizes short conserved sequence stretches within the introns through base pairing between the snRNAs and splice sites, while the protein factors play roles in stabilizing base-paired interactions and mediating structural changes of the spliceosome (for reviews, see references 1 and 2).The spliceosome is assembled by sequential binding of the snRNPs in the order U1, U2, and then the U4/U6.U5 tri-snRNP. Subsequent activation of the spliceosome involves a major structural rearrangement in the spliceosome, leading to the release of U1 and U4 and formation of new base pairs between U2 and U6 and between U6 and the 5= splice site (5=SS) (1-3). The Prp19-associated complex (NTC, for nineteen complex) is required to stabilize the interactions of U6 and U5 with the pre-mRNA after the release of U1 and U4 during formation of the active spliceosome (4). At least eight proteins have been identified as associating with the NTC, and several others have been suggested as putative NTC components (5-10).DEXD/H-box proteins are a family of RNA-dependent ATPases that utilize the energy from ATP hydrolysis to unwind RNA duplexes or to displace proteins from binding to RNA. They function in a wide range of biological processes that involve RNA molecules. Eight DEXD/H-box proteins are required for the splicing process. Each of the catalytic steps requires a DEXD/H-box protein, Prp2 for the first step and Prp16 for the second step, to remodel the spliceosome prior to the catalytic reaction...

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.The splicing of precursor mRNAs (pre-mRNAs) requires five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and numerous protein factors. These factors bind to the premRNA in a sequential manner to form a large ribonucleoprotein complex, called the spliceosome, which catalyzes two consecutive steps of transesterification to excise the intron. After the binding of all five snRNAs, a major structural change occurs on the spliceosome, leading to the release of U1 and U4 and the formation of the active spliceosome, which is competent for catalyzing transesterification reactions (for a review, see references 45 and 47).The spliceosome is a highly dynamic structure and undergoes repetitive remodeling throughout the assembly pathway to rearrange its structure at the expense of ATP (3,23,45). Eight DExD/H-box ATPases are required for the entire splicing process (36). Among them, Prp2 is required for the first catalytic step, and Prp16 is required for the second step. After the spliceosome is activated, the U2 components SF3a and SF3b, which bind to the branch site, are removed in a Prp2-dependent manner (21, 46). The binding of Prp2 to the spliceosome requires Spp2, originally identified as a multicopy suppressor of the prp2-1 mutation (32, 35). Cwc25 is then recruited to the spliceosome to promote the first transesterification reaction (12). The second transesterification reaction is promoted by Prp22, Prp18, and Slu7 but requires the prior action of Prp16 in an ATP-dependent manner. After the reaction is complete, mature mRNA is first released and the spliceosome is then disassembled. Both steps require ATP and the DExD/H-box proteins Prp22 and Prp43, respectively.During the activation of the spliceosome, a protein complex associated with Prp19, known as the NTC (for nineteen complex), is added to the spliceosome after the release of U1 and U4 (37). The NTC plays a role in stabilizing the association of U5 and U6 by specifying base pair interactions between U6 a...