Prp2-mediated protein rearrangements at the catalytic core of the spliceosome as revealed by dcFCCS

Abstract

The compositional and conformational changes during catalytic activation of the spliceosome promoted by the DEAH box ATPase Prp2 are only poorly understood. Here, we show by dual-color fluorescence cross-correlation spectroscopy (dcFCCS) that the binding affinity of several proteins is significantly changed during the Prp2-mediated transition of precatalytic B act spliceosomes to catalytically activated B* spliceosomes from Saccharomyces cerevisiae. During this step, several proteins, including the zinc-finger protein Cwc24, are quantitatively displaced from the B* complex. Consistent with this, we show that Cwc24 is required for step 1 but not for catalysis per se. The U2-associated SF3a and SF3b proteins Prp11 and Cus1 remain bound to the B* spliceosome under near-physiological conditions, but their binding is reduced at high salt. Conversely, high-affinity binding sites are created for Yju2 and Cwc25 during catalytic activation, consistent with their requirement for step 1 catalysis. Our results suggest high cooperativity of multiple Prp2-mediated structural rearrangements at the spliceosome's catalytic core. Moreover, dcFCCS represents a powerful tool ideally suited to study quantitatively spliceosomal protein dynamics in equilibrium.

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

“…In contrast, the majority of Yju2 remained stably associated with the ILS (Table 1). Cwc24 and Cwc27 were not detected in the 35S ILS, consistent with the fact that they dissociate from the spliceosome already during Prp2-mediated catalytic activation of the B act complex (Warkocki et al 2009;Ohrt et al 2012). We note that Cwc23 was not detected by MS either here or in previous studies (Warkocki et al 2009).…”

“…For example, we showed recently that catalytic activation mediated by the RNA helicase Prp2 is accompanied by the almost quantitative dissociation of Cwc24 and Cwc27 from the spliceosome (Ohrt et al 2012). Comparison of the protein composition of post-catalytic spliceosomes and the isolated ILS revealed that only a handful of proteins are displaced upon Prp22 action (Supplemental Fig.…”

“…The anti-Snu114 immunoprecipitates from the pooled fractions 2-4 and 5-7 contained only low amounts of IL and U6 snRNA, (i.e., 1%-3% and ;7% of the respective input RNA). As no antibodies against a U2 protein were available, we performed a similar experiment but with a yeast strain expressing the U2-specific Cus1 protein tagged with GFP (green fluoresent protein) (Ohrt et al 2012). Antibodies against GFP precipitated the U2 snRNP, and not U5, from the pooled gradient fractions (Fig.…”

“…The transition from the B to the B act complex involves a significant change in the spliceosome's protein composition, with numerous polypeptides such as the U1 and U4/U6 proteins being displaced, while ;20 others, including those of the NTC (nineteen complex) are stably recruited to the B act complex (Chan et al 2003;Fabrizio et al 2009;Warkocki et al 2009). For catalytic activation of the spliceosome, the DEAH-box helicase Prp2 binds together with a cofactor, Spp2, and restructures the B act complex in an ATP-dependent manner, yielding the B* complex (Roy et al 1995;Silverman et al 2004); this leads to, among other things, destabilization and/or disruption of the binding of several spliceosomal proteins (Warkocki et al 2009;Lardelli et al 2010;Ohrt et al 2012). Upon recruitment of the step 1 factor Cwc25 to the B* complex, step 1 catalysis occurs, whereby the adenosine at the BS attacks the 59 SS, generating the cleaved exon 1 and intron-lariat (IL)-39 exon intermediates (Chiu et al 2009;Warkocki et al 2009).…”

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…We therefore tested whether Snu17p interacts with pre-mRNA in the context of native spliceosomes. Because RES-complex proteins were previously shown to be most abundant and stably bound in activated yeast spliceosomes (B act ) 26,27 , we analyzed whether Snu17p contacts the pre-mRNA in purified B act complexes. To this end, we generated a yeast strain carrying the prp2-1 mutation as well as a C-terminally tandem affinity purification (TAP)-tagged version of Snu17p.…”