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. 2017 Feb 17;45(3):1539-1552.
doi: 10.1093/nar/gkw1233.

Functional Link Between DEAH/RHA Helicase Prp43 Activation and ATP Base Binding

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Free PMC article

Functional Link Between DEAH/RHA Helicase Prp43 Activation and ATP Base Binding

Julien Robert-Paganin et al. Nucleic Acids Res. .
Free PMC article

Abstract

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.

Figures

Figure 1.
Figure 1.
Prp43 structure. (A) Overall structure of Prp43 in complex with ADP. (B–D) Close up representation of the stacking of the the R- and F- motifs on the ADP nucleotide (B), the CDP nucleotide (C), the ADP nucleotide with F357 in the open conformation modelled from the MLE structure (D). Two orthogonal orientations are shown and the RecA2 domain has been removed for clarity. The figures of the structures are performed with UCSF Chimera (51), ePMV (52) and Blender (http://www.blender.org).
Figure 2.
Figure 2.
NTPase activities: role of Pfa1 and base stacking. Enzymatic assays were performed with BiomolGreen reagent. All conditions were tested three times and the kinetic parameters were determined and represented by non-linear fit to the Michaelis–Menten equation. Comparison of Prp43 WT (A), Prp43–R159A (B) and Prp43–F357A (C) ATPase activity in absence and presence of the G-patch co-factor Pfa1. (D) Comparison of puric and pyrimidic NTPase activities of Prp43. (E, F) CTPase activity of Prp43–R159A and Prp43–F357A in absence and presence of Pfa1.
Figure 3.
Figure 3.
Affinity of Prp43 for ADP and CDP is impaired without base stacking. The affinity of Prp43-WT and Prp43–F357A for NDPs was assessed by ITC and each measure was performed three times. Affinities of Prp43-WT for ADP (A) and CDP (B) and of Prp43–F357A for ADP (C) and CDP (D).
Figure 4.
Figure 4.
Influence of stacking on Prp43 helicase activity. Helicase assays were performed with a 3΄-5΄ substrate (A) which is a RNA–DNA hybrid substrate. The 113 nucleotide-long RNA probe corresponds to the 5΄ stem loop and H box of snR5 snoRNA. It was annealed with a 21 nucleotide-long fluorescently labelled DNA oligonucleotide. Unwinding of a DNA–RNA substrate by Prp43WT (B), Prp43–F357A (C) and Prp43–R159A (D) was assessed following incubation with ATP or CTP and with or without Pfa1 as indicated. Positions of the RNA/labelled DNA substrate and the unwound oligonucleotide are indicated.
Figure 5.
Figure 5.
Prp43 R159 is important for rRNA processing. Strain GAL::PRP43 expressing the chromosomal PRP43 open reading frame under the control of the glucose-repressible promoter was transformed with pHA113 plasmids carrying PRP43-WT (lanes 1 and 2), Prp43–R159A (lane 4), PRP43-E216A (lane 5) or Prp43–F357A (lane 6), or with the empty pHA113 vector as a control (lane 3). The resulting strains were grown on a galactose-containing medium (chromosomal PRP43 expressed) and shifted to glucose for 27 h to deplete endogenous Prp43 proteins. Cells were harvested, total RNAs were extracted and the accumulation levels of the indicated pre-rRNAs were analysed by northern blot using specific oligonucleotide probes. The processing defects observed with PRP43–R159A can be compared to those observed in absence of Gno1 (lane 8, see (28)). The 27SA2 intermediate is detected using two different probes hybridizing either within ITS2 (upper signal, detection of 27SA2 and 27SB intermediates) or within ITS1 between sites A2 and A3 (detection of 27SA2 alone).
Figure 6.
Figure 6.
Sedimentation profiles of snR39, snR41, snR50 and U3 in absence of Gno1. (A) Sedimentation profiles of different snoRNAs: snR39, snR41, snR50 and U3 snoRNA as a reference, using extracts from cells expressing or not the G-patch co-factor Gno1. (B) PhosphorImager quantifications of the pool of free snoRNAs (snoRNAs present in fractions 1–5) relatively to the signal corresponding to the snoRNAs in the input sample (T).

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