The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

Teigelkamp, Stefan; McGarvey, Margaret; Plumpton, Mary; Beggs, Jean D.

doi:10.1002/j.1460-2075.1994.tb06332.x

Cited by 56 publications

(51 citation statements)

References 64 publications

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“…Since PRP2 activity is an essential requirement for the first transesterification reaction (27) PRP2A spliceosomes could not process pre-mRNA into spliced mRNA (Fig. 3A, lanes 3-5), and they formed splicing complex I but no splicing complex 11 (28,29 and data not shown). Complementation with a heat-inactivated temperature-sensitive prp8 extract restored splicing activity (Fig.…”

Section: Methodsmentioning

confidence: 97%

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

1995

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PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase Ti treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branchpoint-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattem of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.

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Section: Methodsmentioning

confidence: 97%

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

1995

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“…It is also unclear which RNA structure(s) in the spliceosome is/are the primary target(s) of Prp2/ATPase activity or whether Prp2 affects protein-protein interactions directly. Prp2 has been cross-linked to the region between the BS and the 39 SS (Teigelkamp et al 1994), raising the possibility that Prp2 modulates interactions between the BS region and the U2 proteins. Alternatively, contact with this stretch of RNA might merely be required to stimulate Prp2/ATPase activity.…”

Section: Cwc24 Functions In the Generation Of An Active Spliceosome Bmentioning

confidence: 99%

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

Ohrt¹,

Prior²,

Dannenberg³

et al. 2012

RNA

132

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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.

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“…Thus, it has been hypothesized that destabilization of SF3 by Prp2p could remove steric constraints and allow Cwc25p binding to enable positioning of the branch site for 5 ′ splice site cleavage (Chiu et al 2009;Warkocki et al 2009;Lardelli et al 2010;Tseng et al 2010;Yeh et al 2010). Since Prp2p cross-links to the pre-mRNA just downstream from the branch site and this region is required for Prp2p function (Teigelkamp et al 1994;Liu and Cheng 2012), Prp2p has been proposed to destabilize directly interactions between the pre-mRNA and SF3 near the branch site of the pre-mRNA (Warkocki et al 2009;Lardelli et al 2010). The suppression of prp2 by prp9-1 ( Fig.…”

Section: The Role Of Prp2p In Destabilizing Proteinsmentioning

confidence: 99%

“…Like many other DExD/H-box ATPases, the ATPase activity of Prp2p is stimulated by RNA binding (Kim et al 1992). Prp2p cross-links to pre-mRNA downstream from the branch site, and this region of the substrate is required during spliceosome activation, so it has been proposed that Prp2p could destabilize interactions involving the pre-mRNA near the branch site (Teigelkamp et al 1994;Liu and Cheng 2012). Indeed, recent studies have begun to elucidate the hallmarks of the role of Prp2p in activation as characterized by protein dynamics at the branch site.…”

Section: Introductionmentioning

confidence: 99%

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

Wlodaver¹,

Staley²

2014

RNA

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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.

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The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA.

Cited by 56 publications

References 64 publications

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing

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

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

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