The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome

Abstract

After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATPdependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNAdependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA-RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5 ′ splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.

The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome

“…For most of the spliceosomal ATPase/helicases, including Prp2, evidence has been provided that they also function by proofreading certain steps of spliceosome assembly (Burgess and Guthrie 1993;Semlow and Staley 2012;Koodathingal and Staley 2013;Wlodaver and Staley 2014). The exact mechanisms by which this is achieved by the various helicases are not always clear; in particular whether they indeed act as molecular timers.…”

“…Prp2 activity also creates a high-affinity binding site for Yju2 and the step 1 factor Cwc25, the binding of which may lead to a closed conformation of the step 1 catalytic center and thus facilitate efficient step 1 catalysis, leading to formation of the spliceosomal complex C (Konarska and Query 2005;Warkocki et al 2009;Ohrt et al 2012;Krishnan et al 2013). Finally, Prp2's activity may also directly or indirectly destabilize RNA elements that comprise the catalytic core, such as the U2/U6 helix I, to promote a fully competent catalytic conformation of the step 1 catalytic center (Wlodaver and Staley 2014).…”

Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes

“…The U5-100K helicase is involved in the switch of U1 for U6 at the 5′ss, which also requires U5-220K/Prp8 [12,30]. During spliceosome activation (B to B* complex transition), Prp2 participates with U5-200K/Brr2 for unwinding U4/U6 [57,64], rearranging the spliceosome and repositioning the substrate [65], probably involving the Brr2 modulator U5-116K/Snu114 GTPase. As expected for splicing superfamily 2 helicases [66], the C-terminus of most Entamoeba DExH/ D-box RNA helicases involved in splicing is conserved (Supplemental Fig.…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…Prp2 is required for the transition from the B act to the B Ã complex, 104 during which it remodels binding of the multimeric SF3b complex to the branch point region, 17,105 reduces association of SF3a and SF3b proteins, 106 leads to displacement of the Cwc24 and Cwc27 components of the 19 complex (NTC) as well as of the Bud13 subunit of the RNA retention and splicing (RES) complex 106 and proofreads the catalytic core of the spliceosome. 107 Consistent with some of these functions, the SF3B1 (Hsh155) protein in the B act complex bridges between the plug domain of Brr2 and Prp2. Brr2 additionally contacts the splicing factors SF3B3 (Res1), Cwc24, Cwc27 and the endonuclease domain of Prp8.…”

“…107,114,[121][122][123][124] As Brr2 may be involved in the recruitment of other spliceosomal remodeling enzymes during various stages of splicing and might additionally influence their activities via protein-protein interactions (see above), it could modulate splicing fidelity and alternative splicing indirectly via recruitment and regulation of these other enzymes. This idea is in line with previous proposals of Brr2 functioning as a landing pad for multiple other helicases, which might sequentially interact in similar fashion with the C-terminal cassette.…”