Characterization of Dominant-negative Mutants of the DEAH-box Splicing Factors Prp22 and Prp16

Susanne Schneider,Hans-Rudolf Hotz,Beate Schwer

doi:10.1074/jbc.m112473200

Abstract

Saccharomyces cerevisiae Prp22 and Prp16 are RNA-dependent ATPases required for pre-mRNA splicing. Both proteins are members of the DEXH-box family of nucleic acid-dependent NTPases. Prior mutational analysis of Prp22 and Prp16 identified residues within conserved motifs I (GXGKT), II (DEAH), and VI (QRXGRXGR) that are required for their biological activity. Nonfunctional Prp22 and Prp16 mutants exerted a dominant negative effect on cell growth. Here we show that overexpression of lethal Prp22 mutants leads to accumulation of unspliced pre-mRNAs and excised introns in vivo. The biochemical basis for the lethality and inhibition of splicing in vivo was determined by purifying and characterizing recombinant mutant proteins. The lethal Prp22 mutants D603A and E604A in motif II and Q804A and R808A in motif VI were defective for ATP hydrolysis and mRNA release from the spliceosome, but were active in promoting step 2 transesterification. Lethal Prp16 mutants G378A and K379A in motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry. The ATPase-defective mutants of Prp16 and Prp22 bound to spliceosomes in vitro and blocked the function of the respective wild-type proteins in trans. Comparing the mutational effects in Prp16 and Prp22 highlights common as well as distinct structural requirements for the ATP-dependent steps in pre-mRNA splicing.

Highlights

Nucleic acid-dependent NTPases of the DEX(H/D)-box family play important roles in many biological processes including transcription, DNA repair, and pre-mRNA splicing
That the ATPase activity of Prp22 was necessary for mRNA release was inferred from an analysis of a mutant in which Lys-512 in motif I was replaced by alanine; the K512A mutant was lethal, defective for ATP hydrolysis and for mRNA release in vitro [15]
Effects of Prp22 Mutations on ATP Hydrolysis—Conserved residues in motifs I (511GKT), II (603DEAH), and VI (804QRKGRAGR) are essential for the biological function of Prp22; alanine substitutions for Lys-512, Asp-603, Glu-604, Gln-804, and Arg-808 are lethal in vivo [18]

Summary

EXPERIMENTAL PROCEDURES

An aliquot of the peak fraction containing recombinant Prp was diluted with buffer IV (20 mM Tris-HCl, pH 7.6, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10% glycerol) to a final NaCl concentration of 30 mM, and loaded on a 1-ml DEAE-Sepharose CL-6B column equilibrated with 30 mM NaCl in buffer IV. Reaction mixtures (30 ␮l) contained 50 mM triethanolamine, pH 8.2, 75 mM potassium acetate, 1 mM dithiothreitol, 1.25 mM MgCl2, 0.5 mM [␥-32P]ATP, 0.25 mM poly(A) (expressed as concentration of adenosine), and increasing amounts of Prp proteins (90 –360 ng) were incubated for 20 min at 23 °C. Reaction mixtures (20 ␮l) containing 40 mM Tris-HCl, pH 8.0, 2 mM dithiothreitol, 2 mM MgCl2, 1 mM [␥-32P]ATP, 0.5 ␮g of poly(A) and Prp proteins as indicated were incubated for 30 min at 30 °C. The blots were stripped by boiling in hot water and re-probed with intron probes (CYH2, ACT1, and SNR17A)

RESULTS

DISCUSSION

Additions and Corrections