Suitability of dual color Fluorescens Cross Correlation Spectroscopy (dcFCCS) for

With the experiments done in this work we demonstrated that dcFCCs is, in combination with the established in vitro reconstitution system of functional spliceosomes, a powerful approach to investigate dynamics of purified spliceosomes.

For dcFCCS measurements, yeast spliceosomes were stalled before step 1 by using the temperature-sensitive mutant prp2-1 yielding a B^actΔPrp2 spliceosome. It was shown by the work of Warkocki et al (2009) that highly purified B^actΔPrp2 can be catalytically activated to form B* when supplemented with recombinant Prp2 and Spp2 splicing factors (Warkocki, Odenwalder et al. 2009). Purified B^actΔPrp2 complexes assembled on actin pre-mRNA labeled at its 5’ end with the red fluorescent dye Atto647N were used. Proteins were labeled in vivo by fusing the protein of interest with a fluorescent protein EGFP (enhanced green fluorescent protein) by genetic modification. As in any study involving the introduction of bulky labels, there was a risk of label-induced impaired behavior. In the system described here, neither the label on the pre-mRNA nor those on the various proteins impaired the activity of the spliceosomes examined. The resulting doubly fluorescent-labeled and purified spliceosomal complexes were analyzed before and after catalytic activation by Prp2 and were found to be stable under our dcFCCS conditions as demonstrated for Snu114-EGFP. Snu114 is known to bind to the spliceosome throughout the splicing cycle (Fabrizio, Laggerbauer et al. 1997) (Fabrizio, Dannenberg et al. 2009) and was therefore used as a ‘‘positive control’’ in our experiments. The dcFCCS results of our complexes were sufficiently reproducible and allowed the investigation of the binding and release of the proteins of interest.

124 4.8 Prp2-mediated transformation of the B^act into the B* complex

substantially alters the binding affinity of several proteins at the catalytic core of the spliceosome

Although the essential role of Prp2 in the catalytic activation of the spliceosome was established earlier, its mechanism of action has remained enigmatic (Kim and Lin (1996). The structural change the spliceosome undergoes upon Prp2-mediated, ATP-dependent activation must be profound, given the substantial change in S value seen between the precursor B^actΔPrp2 complex (45S) and the catalytically activated spliceosome B* (40S) (Kim and Lin (1996); Warkocki, Odenwalder et al. (2009). We expanded our MS data by dcFCCS showing that the binding affinity of at least seven proteins, i.e., the U2 SF3a/b proteins Prp11 and Cus1, the RES complex protein Bud13, Cwc24, Cwc27, Yju2, and Cwc25, is quantitatively and qualitatively altered by Prp2-mediated catalytic activation of the spliceosome. Due to their characteristics in their binding behavior, these proteins can be divided into three groups. The first group comprises Cwc24, Cwc27, and Bud13, which are tightly bound to the B^actΔPrp2 spliceosome and which dissociate almost completely (Cwc24 and Cwc27) or partly (Bud13), even under near-physiological conditions upon Prp2-mediated activation and conversion to the B* complex. The second group includes the SF3a/b proteins Prp11 and Cus1, whose binding to the spliceosome is weakened by the catalytic activation thus becoming salt-sensitive, such that they dissociate from the B* complex at higher salt concentrations, while staying associated with the B* spliceosome at non-stringent conditions (Fig. 3.18). The third group contains proteins whose binding to the spliceosome is enhanced during the B^act to B* transition and includes Yju2 (which is recruited at the stage of the B^act complex and which is more weakly bound prior to catalytic activation) and Cwc25 (which before the transition was not bound at all and is thus recruited to B*). Both proteins are known to be required for the first catalytic step that is catalyzed by complex B*.

125 4.9 Cwc24 functions in the generation of an active spliceosome but is not

required for splicing catalysis per se

There is little known about the function of Cwc24 and Cwc27 in splicing. Cwc27 contains a peptidylprolyl isomerase and also has an evolutionarily conserved counterpart in the human spliceosome, NYCO-10 (Ohi, Link et al. (2002); Fabrizio, Dannenberg et al. (2009), but nevertheless, Cwc27 is not essential for the growth of S.

cerevisiae under standard conditions (Winzeler, Shoemaker et al. (1999),Giaever, Chu et al. (2002). In contrast, Cwc24 is known to be essential for yeast growth, and it is needed for the splicing of the U3 snoRNA precursor in vivo (Goldfeder and Oliveira (2008). Our results that Cwc24 and Cwc27 dissociate almost quantitatively from the spliceosome during or after the Prp2-mediated catalytic activation of the spliceosome (Fig. 3.15) was the first indication that they are not required for catalysis of the splicing reaction per se. This fact led us to investigate the role of Cwc24 in splicing in more detail. The depletion and reconstitution experiments, done in our laboratory, confirmed that the presence of Cwc24 is necessary for splicing to proceed through the first catalytic step in vitro (Ohrt, Prior et al. 2012). Surprisingly, they also showed that Cwc24 is not required during the actual catalysis; catalytically activated B*

complexes that had lost all of their Cwc24 were still able to perform the first catalytic step of splicing efficiently when complemented with the step 1 factor Cwc25 (Fig.

3.14B). Thus, the function of Cwc24 must be to assist in the assembly or the maturation of the activated spliceosome B^act. Consequently, we regard Cwc24 as an assembly factor for the B^act complex that is probably involved in generating the correct structure or conformation of the B^act complex. Therefore it will be interesting to find out which proteins of the B^act complex are direct interaction partners of Cwc24 and whether it also binds to spliceosomal RNAs or the pre-mRNA.

Earlier in this work, we showed that the proteins of the RES complex are recruited to the spliceosome at the time of the B complex and that it is stoichiometrically present in B^act (Table 3.1 and Figure 4.2). Our dcFCCS results demonstrate that the RES protein Bud13 loses its affinity for the B* complex. Previous studies showed that the RES complex is not essential for yeast viability, but is required for efficient splicing in vitro and in vivo. The work by Dziembowski et al. (2004) demonstrated that the inactivation of these proteins causes leakage of unspliced pre-mRNA from the

126 nucleus (Dziembowski, Ventura et al. (2004). Our present results are consistent with the idea that the RES proteins remain associated with the spliceosome until Prp2-mediated catalytic activation of the spliceosome and first step of catalysis to prevent the premature nuclear release of unspliced pre-mRNA.

4.10 Sf3a/b proteins remain bound to the B* spliceosome under