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,

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...

Azaarenes belong to the newly identified classes of anthropogenic pollutants. To our knowledge, this study presents the first result of pollution history of azaarenes and their controlling factors in estuary. To understand the transport mechanisms, surface sediment samples were collected from 18 sites of the Danshuei River during the summer and the winter of 2005. The highest concentrations of azaarene, 21.9 ng g 21 for 2-ring, 9.89 ng g 21 for 3-ring, and 9.17 ng g 21 for 4-ring, were observed in midstream surface sediments near the Shi-Zi-Tou waste water pumping station. Therefore, this waste water pumping station not only provides a good sediment accumulation environment but also discharges small particle size sediments, which contain mass amounts of azaarenes into the Danshuei River. This research also finds highly significant covariations among 3-ring and 4-ring azaarenes, which suggests similar source strengths and transport mechanisms for these compounds. Additionally, core samples were collected from two sites of the Danshuei River and analyzed to determine the pollution history. Values found in the core sediments correspond to pollution events in recent history. Contaminant inputs via in situ burning of dumping field and wastewater discharge for the periods of 1975-1985 and 2004-2005

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