Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function - PubMed (original) (raw)

Mutations in PRP43 that uncouple RNA-dependent NTPase activity and pre-mRNA splicing function

Naoko Tanaka et al. Biochemistry. 2006.

Abstract

Saccharomyces cerevisiae Prp43 is a DEAH-box RNA-dependent ATPase that catalyzes the release of excised lariat intron from the mRNA spliceosome. Previous studies identified mutations in Prp43 motifs I, II, and VI that were lethal in vivo and ablated ATP hydrolysis in vitro. Such Prp43 mutants exerted dominant-negative growth phenotypes when expressed in wild type cells and blocked intron release in vitro when added to yeast splicing extracts. Here, we assessed the effects of alanine and conservative substitutions at conserved residues in motifs Ia ((146)TQPRRVAA(153)), IV ((307)LLFLTG(312)), and V ((376)TNIAETSLT(384)) and thereby identified Arg150 (motif Ia), Phe309 (motif IV), Thr376, Leu383, and Thr384 (motif V) as being important for Prp43 function in vivo. Motif V mutations T376V, T384A, and T384V were lethal and dominant negative in vivo, and the mutant proteins inhibited lariat release in vitro. The T384A and T384V proteins were proficient for ATP hydrolysis, suggesting that ATPase activity is necessary, but not sufficient, for Prp43 function. We report that Prp43 hydrolyzes all common NTPs and dNTPs and unwinds short 5'/3' tailed RNA/DNA duplexes in an ATP-dependent fashion. Optimal ATP hydrolysis requires an RNA cofactor of >or=20 nt. Prp43 is largely indifferent to mutations in its C-terminal segment, which is conserved in the DEAH-box splicing factors Prp2, Prp16, and Prp22.

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Figures

Figure 1

Figure 1

Mutational analysis of motifs Ia, IV and V. The amino acid sequences of Prp43 motifs Ia, IV and V are shown at the top. The numbers refer to the residues in Prp43, the positions that were replaced by alanine are underlined. (A) Summary of mutational effects. The ability of the various PRP43 alleles to complement a _prp43_Δ strain was tested by plasmid shuffle. Alleles that did not complement the _prp43_Δ strain under 5-FOA selection are indicate by ‘lethal’. Viable mutants were plated to rich medium (YPD) and incubated at 14, 25, 30 and 37°C. Growth was scored based on colony size. +++ indicates growth comparable to wild type Prp43; + denotes slow growth at all temperature; cs and ts indicate a conditional growth phenotype at 14 and 37°C respectively. (B) _prp43_Δ cells carrying wild type PRP43 (WT) or the indicated mutants were grown in liquid medium at 30°C. The _A_600 was adjusted to 0.1 and aliquots (3 _μ_l) of serial 10-fold dilutions were spotted onto YPD agar medium. The plates were photographed after incubation for 2 days at 37°C, 3 days at 25°C and 6 days at 14°C.

Figure 2

Figure 2

RNA-dependent ATPase activity. (A) Prp43 proteins (1 _μ_g) were separated by 8% SDS-PAGE and visualized by Coomassie staining. The positions and sizes (in kilodaltons) of marker proteins are indicated at the left. (B) Pi release was measured at 1 mM ATP-Mg and 0-60 _μ_M of poly(A) (measured as AMP concentration). ATP hydrolysis (min-1) of wild type Prp43 (WT) (◆); T384S (Δ); T384A (○); T376S (▲); T384V (□); T376A (●); T376V (■) was plotted as a function of poly(A) concentration. Each datum represents the average of two measurements, the error bars indicate the deviation from the average. (C) Summary of the ATPase activities. (-RNA): ATP hydrolysis (min-1) was determined for the Prp43 proteins (50 nM) during 60 min incubations in the presence of RNase A. (+RNA): _k_cat values were determined from Lineweaver-Burk plots of the curves shown in (B) using the EnzymeKinetics Program (Trinity Software). The _K_m values for poly(A) RNA was determined from the Lineweaver-Burk plot of the data shown in (B). ATP hydrolysis of T376V were measured at 0-500 _μ_M poly(A).

Figure 3

Figure 3

Effect of RNA length. (A) ATPase activity (min-1) was measured as a function of 0-2 _μ_M of homo-oligomers of 40 (○), 30 (□) and 20 (Δ) adenylates. The concentrations of Prp43 and ATP-Mg were 50 nM and 1 mM, respectively. The inset shows ATP hydrolysis as a function of 0-10 _μ_M (A)10. Each datum represents the average of two measurements, the error bars indicate the deviation from the average. (B) ATP hydrolysis was measured at 50 nM Prp43 and 1 mM ATP-Mg in the presence of increasing concentrations (0-500 nM) of mixed RNAs of 40 (●), 30 (■) and 20 (▲) nucleotides in length. (C) Summary. _k_cat (min-1) and _K_m values were determined from Lineweaver-Burk plots of the data shown in (A) and (B).

Figure 4

Figure 4

RNA40-stimulated ATP hydrolysis by Prp43 mutants. (A) ATP hydrolysis at 1 mM ATP-Mg was measured as a function of increasing concentrations (0-200 _μ_M) of RNA40 for T384S (Δ), T376A (●), T376S (▲) and T384A (○). Each datum represents the average of two measurements, the error bars indicate the deviation from the average. (B) _k_cat (min-1) and _K_m values for RNA40 were determined from Lineweaver-Burk plots of the data shown in (A).

Figure 5

Figure 5

RNA binding. Reaction mixtures containing the indicated 32P-labeled RNA and increasing concentrations (0.5, 1, 2, 5, 10, 20, 40, 60, 80, 100, 200, 500 and 1000 nM) of Prp43 were incubated for 30 min at 30°C. Protein was omitted in the first lane (-) of each panel. The products were analyzed by native PAGE. Autoradiograms of the dried gels are shown. The positions for free RNA and stable RNA-protein complexes (*) are indicated. The bar denotes complexes that possibly dissociate during electrophoresis.

Figure 6

Figure 6

RNA binding. (A) 32P-labeled RNA40 and RNA30 was incubated with increasing concentrations of Prp43 (0.5, 1, 2, 5, 10, 20, 50 and 100 nM) and analyzed by native PAGE. (B) 32P-labeled RNA40 was incubated with 1, 10 and 100 nM of the indicated mutant proteins and the products were analyzed by native PAGE. Autoradiograms of the dried gels are shown.

Figure 7

Figure 7

Unwinding activity. (A) The helicase substrate, in which a 99 nt RNA strand is annealed to a 20 nt 32P-labeled DNA oligonucleotide is depicted at the top. ΔT shows migration of the labeled DNA oligonucleotide after heating of the substrate for 3 min at 95°C. Left panel: Reaction mixtures containing 1 _μ_M Prp43 were supplemented (+) with 0.5 mM ATP-Mg (ATP) or 2 mM AMPPCP-Mg (PCP), incubated for 1 h at 37°C and analyzed by native PAGE; (-) indicates that ATP-Mg or AMPPCP-Mg was omitted. Right panel: Reaction mixture contained 1 mM ATP-Mg, 1.25 nM helicase substrate and 1 _μ_M Prp43. Aliquots were withdrawn at the indicated times and halted by addition of SDS-containing loading buffer and transfer to ice. The products were analyzed by native PAGE and autoradiograms of the dried gels are shown. (B) Unwinding of a 5′ tailed 30 bp duplex (left panel), and a 3′ tailed 30 bp helicase substrate (right panel) as a function of time. Reaction mixtures contained 1 mM ATP-Mg, 1 _μ_M of wild type Prp43 and 1.25 nM helicase substrate depicted above each panel. (C) Unwinding by mutant proteins. Reaction mixtures containing 1.25 nM 5′/3′ tailed helicase substrate (depicted in 7A), 1 mM ATP-Mg and 1 _μ_M of wild type Prp43 (WT) or the indicated mutant protein, were incubated for 1 h at 37°C. The products were analyzed by native PAGE and the amounts of 32P-labeled helicase substrate (ds) and single-stranded product (ss) were quantified using a phosphorimager. % unwinding was calculated and the background (incubation in the absence of protein) was deducted in every case. Each datum is the average of two experiments. (D) Prp43 hydrolyzes all common NTPs and dNTPs. Reaction mixtures (100 _μ_l) contained 40 mM Tris-HCl (pH 8), 2 mM DTT, 1 mM NTP-Mg, 0.6 mM poly(A) and 10 nM Prp43. Pi release was measured after incubation for 2-10 min at 30°C; the values are averages from two measurements.

Figure 8

Figure 8

(A) Growth inhibition caused by overexpression of lethal Prp43 mutants. Wild type PRP43 and the indicated mutant alleles under the control of the GAL1 promoter were transformed into wild type PRP43 cells. Transformants were grown in liquid cultures (SD-Trp, 2% raffinose). The cultures were adjusted to _A_600 of 10-1, 10-2, 10-3 and 10-4, and 5 _μ_l was spotted to plates containing glucose or galactose (2%) in SD-Trp medium. The plates were photographed after 3 days of incubation at 30°C. (B) In vitro splicing. Reaction mixtures (10 _μ_l) contained 40% whole cell extract, ∼50 fmol of [32P]GMP-labeled actin precursor RNA, 60 mM potassium phosphate, 2.5 mM MgCl2, 2 mM ATP and 100 nM of the indicated protein. A control reaction in which buffer and no exogenous Prp43 protein was added is shown in lane (-). The reaction products were resolved by denaturing PAGE and visualized by autoradiography. The symbols at the left indicate the positions of the following labeled RNA species, proceeding from the top to bottom of the gel: lariat-exon 2 intermediate, lariat-intron, pre-mRNA substrate, mRNA.

Figure 9

Figure 9

Mutational analysis of Prp43. The sequences of the C-terminal segments beginning at motif VI for Prp43, Prp22, Prp16 and Prp2 are aligned. Amino acids that are identical or similar between the four proteins are indicated by ˆ below the alignment. The 97 residues that were replaced by alanines in Prp43 are highlighted in grey. A line above the sequence indicates that clusters of 2 or 3 residues were replaced by alanines; (+) denotes residues at which alanine replacement elicited no phenotype; (*) marks positions at which alanine replacement resulted in a temperature sensitive phenotype at 37°C; (cs) denotes the 2 vicinal residues at which alanine substitutions caused a growth phenotype at 14°C. Table: The ability of the mutated PRP43 alleles to complement a _prp43_Δ strain was tested at 30°C. Growth of the mutant strains was compared to that of wild type PRP43 cells on rich medium at 19, 30 and 37°C. (+++) indicates that the mutant cells grew as well as wild type PRP43 at all temperatures tested; (+) denotes a mutant that grew more slowly than wild type cells; (cs) indicates that the mutant did not form colonies at 14°C; (ts) indicates that the mutant did not form colonies at 37°C; (ts*) means that the mutant formed pinpoint colonies at 37°C; (lethal) indicates mutants that did not grow at 19, 30 or 37°C under 5-FOA selection. An 11-aa segment that is important for Prp2's interaction with the spliceosome is underlined (30). The C-terminal margin for Prp43 activity is indicated by an arrowhead and the bracket denotes a 10-aa segment that is important for activity (18).

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