Splicing Factor Cwc22 Is Required for the Function of Prp2 and for the Spliceosome To Escape from a Futile Pathway

Abstract

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...

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

“…S7). Cwc22 is an integral protein of the B act complex and is required for the catalytic activation of the spliceosome Yeh et al 2011). As Cwc22 remains stably associated with the spliceosome during its catalytic phases and only leaves upon displacement of the mRNA, it could conceivably play a part in stabilizing the binding of the mRNA to the spliceosome.…”

Graded requirement for the spliceosome in cell cycle progression

“…33 Our screen revealed that siRNAs against several spliceosome components resulted in increased cell survival in the presence of taxol. 10 The spliceosome components uncovered in our screen included: SNRPB (SmB/B'), one of 7 core structural spliceosome proteins that directly assemble on the U-rich snRNAs; 34 P14 (SF3B14), a protein that directly contacts the adenosine at the branch point and carries out the first transesterification reaction during splicing; 35 as well as the spliceosome associated components CWC22 36 and CRNKL1. 37 To understand how depletion of these spliceosome components increased cell survival in taxol, we transfected HeLa cells with a pool of 4 siRNAs designed against each of the indicated spliceosome components.…”

Dramatically reduced spliceosome in Cyanidioschyzon merolae

“…Prp2 is at the center of a network of interactions, directly contacting the Sf3 protein Ysf3 (39) as well as Cwc22 and Spp2. Cwc22 is essential for Prp2 function: In its absence, hydrolysis of ATP by Prp2 simply results in the release of Prp2 without promoting the first chemical step (40). Spp2 interacts directly with Prp2 and is released upon ATP hydrolysis (41).…”