ATP-Dependent Remodeling of the Spliceosome: Intragenic Suppressors of Release-Defective Mutants of Saccharomyces cerevisiae Prp22

Corresponding author: Beate Schwer, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021., bschwer{at}mail.med.cornell.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The essential splicing factor Prp22 is a DEAH-box helicase that catalyzes the release of mRNA from the spliceosome. ATP hydrolysis by Prp22 is necessary but not sufficient for spliceosome disassembly. Previous work showed that mutations in motif III (⁶³⁵SAT⁶³⁷) of Prp22 that uncouple ATP hydrolysis from spliceosome disassembly lead to severe cold-sensitive (cs) growth defects and to impaired RNA unwinding activity in vitro. The cs phenotype of S635A (⁶³⁵AAT) can be suppressed by intragenic mutations that restore RNA unwinding. We now report the isolation and characterization of new intragenic mutations that suppress the cold-sensitive growth phenotypes of the T637A motif III mutation (SAA), the H606A mutation in the DEAH-box (DEAA), and the R805A mutation in motif VI (⁸⁰⁴QAKGRAGR⁸¹¹). Whereas the T637A and H606A proteins are deficient in releasing mRNA from the spliceosome at nonpermissive temperature in vitro, the suppressor proteins have recovered mRNA release activity. To address the mechanisms of suppression, we tested ATPase and helicase activities of Prp22 suppressor mutant proteins and found that the ability to unwind a 25-bp RNA duplex was not restored in every case. This finding suggests that release of mRNA from the spliceosome is less demanding than unwinding of a 25-bp duplex RNA; the latter reaction presumably reflects the result of several successive cycles of ATP binding, hydrolysis, and unwinding. Increasing the reaction temperature allows H606A and T637A to effect mRNA release in vitro, but does not restore RNA unwinding by T637A.

THE Saccharomyces cerevisiae PRP22 gene encodes an essential ATPase that functions at two distinct stages during the pre-mRNA splicing pathway. Prp22 is important for the second transesterification step, which entails cleavage at the 3' splice site and exon ligation, and it then catalyzes the release of mRNA from the spliceosome. Whereas the step 2 function of Prp22 is ATP independent, its role in spliceosome disassembly requires ATP hydrolysis (SCHWER and GROSS 1998 ; WAGNER et al. 1998 ).

Prp22 is a member of the DExH-box family of nucleic acid-dependent phosphohydrolases and NTP-dependent helicases (COMPANY et al. 1991 ). DExH proteins play important roles in nucleic acid transactions, including transcription, DNA repair and recombination, and pre-mRNA splicing (DE LA CRUZ et al. 1999 ). The distinctive feature of this family of enzymes is the presence of six conserved sequence motifs arrayed collinearly (GORBALENYA et al. 1989 ; JANKOWSKY and JANKOWSKY 2000 ). Mutational analyses of several DExH-box ATPases, including Prp22, have shown that the conserved residues are important for the enzymatic activities and likely comprise the active site for NTP hydrolysis (GROSS and SHUMAN 1995 , GROSS and SHUMAN 1996 ; HEILEK and PETERSON 1997 ; KIM et al. 1997 ; LEE et al. 1997 ; SCHWER and GROSS 1998 ; WAGNER et al. 1998 ; HALL and MATSON 1999 ; MARTINS et al. 1999 ). In Prp22, alanine substitutions in conserved residues in motifs I (GETGSGKT⁵¹³), II (DEAH⁶⁰⁶), III (SAT⁶³⁷), and VI (QRKGRAGR⁸¹¹) cause lethality or else result in severe growth defects (SCHWER and GROSS 1998 ; WAGNER et al. 1998 ; SCHWER and MESZAROS 2000 ).

The ability of Prp22 to hydrolyze ATP is necessary but not sufficient for Prp22's function in pre-mRNA splicing. Previous work showed that the hydroxyamino acids Ser635 and Thr637 in motif III (SAT) are important for coupling ATP hydrolysis to spliceosome disassembly and to RNA unwinding in vitro. The S635A mutation causes inviability at 30° and results in slow growth at 37°. A genetic screen for revertants of the growth defect of S635A at 30° led to the identification of intragenic suppressor mutations, all of which mapped within the ATPase domain of Prp22, which extends from residues 466–1145 (SCHNEIDER and SCHWER 2001 ). One of the suppressor mutations, S27 (S635A-V539I), restored growth at 30° and coordinately revived spliceosome disassembly in vitro, and it also restored the coupling of ATP hydrolysis to RNA unwinding (SCHWER and MESZAROS 2000 ).

The crystal structure of the DExH-box RNA helicase NS3 of hepatitis C virus (HCV) shows that motif III is located in a region that connects globular domain 1, which contains the GKT (motif I) and DExH (motif II) elements, to a second globular domain containing motif VI (QxxGRxxR; YAO et al. 1997 ; KIM et al. 1998 ). KIM et al. 1998 surmised from their crystal structure of NS3 that the conserved side chains in motifs I and II of domain 1 coordinate the divalent cation and contact the ß-phosphate of ATP, whereas the motif VI arginines of domain 2 contact the - and -phosphates of ATP, thereby bridging the two domains together in the ATP-bound state. They suggested that ATP hydrolysis leads to a conformational change that opens the cleft between the two domains and translocates the protein in a 3' to 5' direction along the polynucleotide.

This model invokes flexibility of the hinge region connecting the two domains, which may be impaired in the Prp22 mutant proteins S635A and T637A. Intragenic suppressor mutations likely identify structural elements that either interact with motif III or facilitate conformational steps required for Prp22's function in releasing mature RNA from the spliceosome. Such a conformational step can be measured as unwinding of an RNA duplex, and the V539I-suppressing mutation in S27 (S635A-V539I) did partially restore the ability to unwind a 25-bp RNA duplex to the S635A mutant protein (SCHWER and MESZAROS 2000 ).

To further illuminate the correlation between the mRNA release function of Prp22 and its RNA helicase activity, we performed genetic screens for spontaneous intragenic suppressors of the cold-sensitive prp22-H606A, prp22-T637A, and prp22-R805A mutants. A pseudorevertant within motif III (SAA SAS) and single-amino-acid changes at nine positions restored growth of prp22-T637A at the restrictive temperatures. All but two suppressor mutations of T637A mapped within the ATPase domain. Notably, some of the T637A suppressors and both of the H606A suppressor mutations mapped to residues that were mutated in suppressors of prp22-S635A. Biochemical analyses of three of the suppressor proteins revealed that they restored mRNA release activity to the T637A and H606A proteins. However, in one of the suppressor mutants, RNA unwinding activity was not revived, arguing that the helicase activity measured in vitro does not correspond strictly to the activity needed for spliceosome disassembly.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of spontaneous suppressor mutations:
The mutant strains T637A, H606A, and R805A (MATa ura3-52 trp1-63 his3-200 leu21 ade2-101 lys2-801 prp22::LEU2) contain the prp22-T637A, the prp22-H606A, or the prp22-R805A allele on CEN TRP1 plasmids (SCHWER and MESZAROS 2000 ). Spontaneous suppressor mutations were selected by inoculation of 80 (T637A and R805A) or 120 (H606A) independent cultures from single colonies. After 4–6 days of incubation at 23° (T637A) or 19° (H606A and R805A), aliquots of the liquid cultures were plated to YPD agar at the restrictive temperatures. A single isolate was selected from those cultures that yielded colonies. These were named T1, T2, T3, ... , T13 for T637A; H1 and H2 for H606A; and R1, R2, R3, R4 for R805A. The TRP1 plasmids were isolated and amplified in bacteria. To test whether the suppressor mutation was plasmid linked, the TRP1 CEN plasmids were transformed into a prp22 strain carrying p360-Prp22 (URA3 CEN PRP22). Trp⁺ transformants were selected and streaked onto agar medium containing 0.75 mg/ml 5-fluoroorotic acid (5-FOA) to select against the p360-Prp22 plasmid. The plates were incubated at 34°, a permissive temperature for all strains. Survivors resistant to 5-FOA were streaked to YPD medium and their growth at 37°, 34°, 30°, 25°, 19°, and 14° was compared to strains carrying wild-type PRP22 or the cs mutant alleles prp22-T637A, prp22-H606A, and prp22-R805A. We surmised that suppressor mutations in strains that grew better than the original cold-sensitive (cs) mutant were plasmid linked and due to changes within the PRP22 gene. Serial 10-fold dilutions of the intragenic suppressor mutants were spotted to YPD agar and incubated at 37°, 30°, 25°, and 14° (Fig 2). The complete nucleotide sequences were determined for each of the plasmid-linked prp22-T, prp22-H, and prp22-R alleles.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Intragenic suppressor mutations of H606A and T637A. The coding changes in 15 independent intragenic suppressors of the cold-sensitive growth phenotype of H606A, T637A, and R805A are listed on the left and highlighted above the amino acid sequence of Prp22 on the right. Note that each suppressor, except the pseudorevertant T8 (SAA SAS, indicated by an asterisk) also contains the original Ala substitution. Motifs I–IV are highlighted by shaded boxes and the arrowheads indicate positions 606, 637, and 805 in the Prp22 protein that were replaced by alanines in the original cs mutants.

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Intragenic suppression of cold-sensitive growth. prp22 cells carrying wild-type PRP22, H606A, T637A, R805A, or the indicated suppressors were grown in liquid medium at 30°. The A600 was adjusted to 0.05. Aliquots (3 µl) of serial 10-fold dilutions were spotted on YPD agar medium. The plates were photographed after incubation for 2 days at 37° and 30°, 3 days at 25°, and 7 days at 14°.

Expression and purification of recombinant Prp22 proteins:
Plasmid pET16b-Prp22 encodes an N-terminal His-tagged version of wild-type Prp22 under the control of a T7 promoter (SCHWER and GROSS 1998 ). Here we constructed pET-based plasmids for expression of His-tagged Prp22 missense mutants H606A and R805A and the intragenic suppressor mutants H1 (H606A-A760V), H2 (H606A-A623P), T4 (T637A-M853I), T7 (T637A-G813S), R1 (R805A-V730F), and R2 (R805A-S736I). The expression plasmids were transformed into Escherichia coli strain BL21-Codon Plus (DE3)RIL (Stratagene, La Jolla, CA). Cultures were inoculated from single colonies of freshly transformed cells in Luria-Bertani medium containing 0.1 mg/ml ampicillin. The cultures were maintained in logarithmic growth at 37° to a final volume of 1 liter. When the A₆₀₀ reached 0.6–0.8, the cultures were chilled on ice for 30 min and then adjusted to 0.4 mM isopropyl-ß-thiogalactopyranoside (IPTG) and 2% ethanol. The cultures were then incubated for 16 hr at 17° with constant shaking. Cells were harvested by centrifugation and the pellets were stored at -80°.

The His-tagged Prp22 proteins were purified by Ni-agarose and phosphocellulose column chromatography as described (SCHWER and GROSS 1998 ; SCHWER and MESZAROS 2000 ). Aliquots of the phosphocellulose protein preparations were applied to 4.8-ml 15–30% glycerol gradients containing 250 mM NaCl, 50 mM Tris-HCl (pH 8.0), 2 mM DTT, 1 mM EDTA, 0.1% Triton X-100. The gradients were centrifuged at 4° for 18.5 hr at 47,000 rpm in a Sorvall SW50 rotor. Fractions (0.18 ml) were collected from the tops of the tubes. The Prp22 elution profiles were gauged by SDS-PAGE. Protein concentrations were determined by using the Bio-Rad (Richmond, CA) dye-binding reagent with bovine serum albumin as the standard.

ATPase assay:
Reaction mixtures (20 µl) containing 40 mM Tris-HCl (pH 8.0), 2 mM DTT, 2 mM MgCl₂, 1 mM [-³²P]ATP, 0.5 µg of poly(A), and Prp22 as specified were incubated for 30 min at 30°. The reactions were stopped by the addition of 280 µl of a 5% (w/v) suspension of activated charcoal in 20 mM phosphoric acid. The samples were incubated on ice for 10 min and the charcoal was recovered by centrifugation. ³²P radioactivity in the supernatant was quantified by liquid scintillation counting. The results are average values from duplicate reaction mixes.

In vitro pre-mRNA splicing:
Yeast whole-cell extract from strain BJ2168 was prepared, using the liquid nitrogen method (ANSARI and SCHWER 1995 ). The extract was immunodepleted of Prp22, using Prp22 affinity-purified polyclonal antibodies as described (SCHWER and GROSS 1998 ; SCHWER and MESZAROS 2000). Splicing reaction mixtures (100 µl) contained 50% Prp22-depleted extract, 400 fmol of ³²P-GMP-labeled actin precursor RNA, 60 mM potassium phosphate, 2.5 mM MgCl₂, 2 mM ATP, and 3% PEG₈₀₀₀. The reaction mixtures were incubated for 10 min at 23°, and then 50–100 ng of wild-type or mutant Prp22 (glycerol gradient fractions) was added and incubation was continued for 15 min at either 23° or 30°. The reaction mixtures were halted by transfer to ice. Aliquots (90 µl) were layered onto 15–40% glycerol gradients containing 20 mM HEPES (pH 6.5), 100 mM KCl, 2 mM MgCl₂. The gradients were centrifuged at 4° for 12 hr at 35,000 rpm in a Sorvall TH641 rotor. Fractions (400 µl) were collected from the tops of the tubes. RNA was recovered from the gradient fractions by phenol extraction and ethanol precipitation. RNAs from alternate fractions were analyzed by electrophoresis through a 6% polyacrylamide (19:1) gel containing 7 M urea in TBE (89 mM Tris-borate, 2 mM EDTA). Radiolabeled RNA was visualized by autoradiographic exposure of the dried gel. The amounts of the mRNA products were quantified by scanning the gels using a STORM phosphorimager.

RNA unwinding assay:
Radiolabeled dsRNA helicase substrate was prepared as described previously (LEE and HURWITZ 1992 ; GROSS and SHUMAN 1995 ). The 3'-tailed RNA substrate contained a 25-bp duplex and 3' single-stranded regions of 82 and 78 nucleotides (LEE and HURWITZ 1992 ). The helicase assays were performed as described previously (SCHWER and GROSS 1998 ) and the extent of unwinding after 30 min of incubation at 37° was quantified by scanning the gel using a STORM phosphorimager.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Intragenic missense mutations suppress the cold-sensitive growth phenotype of prp22-T637A, prp22-H606A, and prp22-R805A:
The mutant strains T637A, H606A, and R805A fail to grow at temperatures below 30° (T637A) or 20° (H606A, R805A). Previous work showed that the T637A protein is defective in catalyzing the release of mRNA from the spliceosome in vitro (SCHWER and MESZAROS 2000 ). The inability of T637A to catalyze spliceosome disassembly was not due to a loss in ATPase activity, but rather to a defect in coupling of the energy from ATP hydrolysis to an event that can be measured as RNA unwinding in vitro. To explore genetically the question of how Prp22 harnesses chemical energy to release mRNA, we sought to isolate suppressors of the conditional growth defect of the T637A, H606A, and R805A strains, with the expectation of identifying residues within Prp22 that impact on spliceosome disassembly.

Spontaneous suppressors were selected by growing the T637A, H606A, and R805A mutants at their respective restrictive temperatures (MATERIALS AND METHODS). We determined the intragenic mutations in each independent isolate of the prp22-T, prp22-H, and prp22-R suppressor mutants and found that suppression was due to a single coding change in every case (Fig 1). Growth of the suppressor mutants was compared to that of the wild-type PRP22 and the T637A, H606A, and R805A cells on rich medium (YPD) at 37°, 30°, 25°, and 14° (Fig 2).

Ten intragenic suppressors of the cs phenotype of T637A were selected. These were T2 (T637A-N803K), T4 (T637A-M853I), T5 (T637A-V633F), T6 (T637A-Q585H), T7 (T637A-G813S), T8 (T637S), T9 (T637A-H410P), T10 (T637A-G813S), T12 (T637A-A623D), and T13 (T637A-S399P). These strains all grew at 25° (the restrictive temperature for T637A) and several of the suppressor mutations (T2, T4, T7, and T8) even restored growth at 14° (Fig 2). The spontaneous suppressor mutations in H1 (H606A-A760V) and H2 (H606A-A623P) restored growth of H606A cells at 14° (Fig 2). The intragenic suppressor mutations in R1 (R805A-V730F) and R2 (R805A-S736I) restored growth of R805A cells at 14° (Fig 2).

Of the 15 intragenic suppressors, none entailed reversion of Ala637 to Thr, Ala606 to His, or Ala805 to Arg (Fig 1). However, one of the suppressors, prp22-T8, was a pseudorevertant in which Ala637 was changed to Ser. This result underscores the importance of both of the side-chain hydroxyls in motif III (SAT); the necessity for a Ser or Thr at position 635 in the Prp22 protein has been reported previously (SCHWER and MESZAROS 2000 ).

In 13 of the 15 suppressors (T2, T4, T5, T6, T7, T8, T10, T12, H1, H2, R1, R2, and R4) the mutations were located within the ATPase/helicase domain of Prp22 (spanning amino acids 465–1145; SCHNEIDER and SCHWER 2001 ). In contrast, the T13 (T637A-S399P) and T9 (T637A-H410P) suppressors mapped within the N-terminal domain (Fig 1), which is not required for ATPase activity, but is essential for the in vivo function of Prp22 (SCHNEIDER and SCHWER 2001 ). This finding suggests that the N-terminal portion of Prp22 may influence the activity of the catalytic domain.

The location of the suppressing mutations within the catalytic domain of Prp22 is instructive with respect to internal domain dynamics. Three independent suppressor mutations, T2 (N803K) and T7/T10 (G813S), mapped close to motif VI (⁸⁰⁴QRKGRAGR⁸¹¹; Fig 1). Motif VI residues Gln804, Gly807, Arg808, and Arg811 are essential for the in vivo function of Prp22, for spliceosome disassembly, and for ATP hydrolysis (SCHWER and MESZAROS 2000 ; S. SCHNEIDER and B. SCHWER, unpublished data). Remarkably, five independent isolates of S635A suppressors also contained missense mutations within and proximal to motif VI (SCHWER and MESZAROS 2000 ). These were S15 (S635A-A809S), S19 (S635A-A809S), S13 (S635A-K816E), S22 (S635A-K816E), and S34 (S635A-K816E). We surmise that these changes affect ATP binding and/or conformational changes within Prp22 elicited by ATP hydrolysis.

Certain residues within Prp22 were changed multiple times in different intragenic suppressor mutants. For example, Ala623, located in the segment between the DEAH⁶⁰⁶ box and motif III, was changed to Asp in the T12 suppressor (T637A-A623D) and to Pro in the H2 suppressor (H606A-A623P; Fig 1). These same changes at Ala623 were found previously in the S16 (S635A-A623P) and the S32 (S635A-A623D) suppressors (SCHWER and MESZAROS 2000 ). Thus, replacement of Ala623 by either Pro or Asp can restore growth of three different prp22-cs mutants (S635A, T637A, and H606A). Ala760, which is situated within motif V (⁷⁵⁷TNIAETSLTI⁷⁶⁶), was replaced by Val in H1 (H606A-A760V) and also in S3 (S635A-A760V).

None of the second-site mutations identified in R805A suppressors R1 (R805A-V730F), R2 (R805A-S736I), or R4 (R805A-V730F; Fig 1) had been found in any suppressor of S635A, T637A, or H606A, suggesting that the basis for cold sensitivity and the mechanism for suppression may be distinct for the R805A mutants.

Suppressor mutations restore spliceosome disassembly in vitro:
The suppressor proteins T4 (T637A-M853I), T7 (T637A-G813S), H1 (H606A-A760V), H2 (H606A-A623P), R1 (R805A-V730F), and R2 (R805A-S736I) were produced in bacteria as His-tagged fusions along with Prp22, T637A, H606A, and R805A. The T4, T7, H1, T637A, H606A, and Prp22 proteins were purified from soluble bacterial lysates by Ni-agarose and phosphocellulose chromatography followed by glycerol gradient sedimentation. SDS/PAGE analysis of the peak glycerol gradient fractions showed that the protein preparations were substantially pure (Fig 3). The H2, R1, R2, and R805A polypeptides were recovered exclusively in the insoluble fraction and were therefore not amenable to further analysis.

View larger version (74K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Protein purification. Aliquots (0.75 µg) of the glycerol gradient preparations of wild-type (WT) Prp22; the Ala-substitution mutants T637A and H606A; and the suppressor mutants T4, T7, and H1 were analyzed by electrophoresis on an 8% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie Blue. The asterisk marks the position of Prp22 proteins. The positions and sizes (in kilodaltons) of marker proteins are indicated on the left.

The cs phenotype of the T637A mutant correlates with the failure of the T637A protein to catalyze spliceosome disassembly in vitro (SCHWER and MESZAROS 2000 ). Here we tested spliceosome disassembly by the suppressor proteins T4 and T7 (Fig 4A). Yeast whole-cell extracts depleted of Prp22 were reacted with radiolabeled ACT1 pre-mRNA for 10 min at 23°. The reaction mixtures were then supplemented with purified recombinant wild-type Prp22, T637A, T4, or T7 proteins, and the incubation was continued for 15 min at 23°. The splicing reaction products were analyzed by zonal velocity sedimentation through 15–40% glycerol gradients (Fig 4A). When the Prp22-depleted splicing reactions were reconstituted with Prp22, mature ACT1 mRNA sedimented as a discrete peak near the top of the gradient. As previously reported (SCHWER and MESZAROS 2000 ), in the reactions supplemented with T637A, most of the mRNA was retained in the heavy spliceosome fractions; only 11% of the ACT1 mRNA was released and retrieved in the lighter fractions. In splicing reactions reconstituted with the T4 and T7 suppressor proteins, the amount of released mRNA increased to 44 and 80%, respectively (Fig 4A). These findings suggest that the missense mutations M853I in T4 and G813S in T7 suppress the cs growth defect of the T637A mutant by restoring spliceosome disassembly.

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Motif II and III mutations affect Prp22-catalyzed release of spliced mRNA. Splicing reactions were performed in Prp22-depleted extract at 23° for 15 min. Reaction mixtures were then supplemented with the indicated proteins. (A and B) Reconstitution was carried out at 23°; (C) reactions were supplemented with T637A and H606A proteins at 30°. Incubations were continued for 10 min and the reaction mixtures were analyzed by zonal velocity sedimentation. RNA was isolated from odd fractions (3–29) of each glycerol gradient, analyzed by denaturing PAGE, and visualized by autoradiography. The positions of the lariat-intermediate, lariat-intron, pre-mRNA, and spliced mRNA are indicated at the left of the gels. The levels of mRNA in each lane were quantitated using the STORM phosphorimager.

We also assayed the activity of the H606A mutant and the H1 (H606A-A760V) suppressor protein (Fig 4B). Whereas in reactions reconstituted with H606A 26% of the mature RNA was released, the H1 protein released 79% of mature RNA. Thus, the A760V change elicited a gain of function in spliceosome disassembly.

Release of mature RNA from the spliceosome is temperature dependent:
The permissive temperature for growth of the H606A mutant strain is 20°, whereas the T637A mutant does not form colonies at 25°. When the in vitro splicing assays were performed at 23°, H606A released 26% of ACT1 mRNA, whereas T637A released 11% of mRNA (Fig 4A and Fig B). To test whether the reaction temperature influenced spliceosome disassembly, we reconstituted the splicing reactions at 30° (Fig 4C). At 30°, wild-type Prp22 released 94% of mature RNA (not shown) and T637A and H606A released 77 and 85% of ACT1 mRNA, respectively (Fig 4C). Increasing the reaction temperature also further improved mRNA release by T4 from 44% at 23° to 82% at 30° (not shown).

We conclude that (i) the cs growth phenotype of T637A and H606A is likely due to the inability of the proteins to promote spliceosome disassembly at low temperature; (ii) secondary mutations suppress the cs growth defect by restoring spliceosome disassembly at low temperature; and (iii) increased thermal energy per se compensates for the functional defects of the T637A and H606A mutant proteins in spliceosome disassembly in vitro.

ATP hydrolysis by Prp22 mutant proteins:
Prp22 requires ATP hydrolysis to catalyze spliceosome disassembly (SCHWER and GROSS 1998 ; WAGNER et al. 1998 ). Wild-type Prp22, T637A, H606A, and the suppressor proteins T4, T7, and H1 were assayed for ATP hydrolysis in the presence of poly(A). The profiles of ATPase activity across the glycerol gradients, the final step of purification, coincided with the abundance of the purified Prp22 proteins in every case (not shown). ATP hydrolysis in the presence of poly(A) was linear with time for at least 40 min (not shown).

Using the protein preparations shown in Fig 3, we determined that the extent of ATP hydrolysis during a 30-min incubation at 30° was proportional to the amount of input enzyme (Fig 5A). In agreement with previous studies, the activity of the T637A mutant, which hydrolyzed 650 ATP min^-1, was higher than that of wild-type Prp22 (430 min^-1; Fig 5B). Similarly, the H606A and T4 proteins were more active, hydrolyzing 550 and 630 ATP min^-1, respectively. The activities of T7 and H1 (160 and 150 ATP min^-1) were 35% of wild-type Prp22.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 5. The ATPase activity of wild-type Prp22 (WT), T637A, H606A, T4, T7, and H1 was measured in the presence of poly(A) cofactor and (A) is plotted as a function of input Prp22 proteins (in nanograms). (B) The ATPase activities, measured in the absence and presence of poly(A), are expressed as turnover numbers (per minute) for the various Prp22 mutant variants.

ATP hydrolysis by Prp22 was stimulated 11-fold by poly(A) (Fig 5B). When we tested the extent to which the mutant proteins T637A, T4, T7, H606A, and H1 were stimulated by the presence of poly(A), we found that the basal levels of ATP hydrolysis by T637A, T4, and T7 were in a similar range as wild-type Prp22 (40–60 ATP min^-1). However, the basal ATPase activities of H606A and H1 were 4-fold higher, such that in the absence of poly(A), H606A and H1 hydrolyzed 160 and 190 ATP min^-1, respectively (Fig 5B). These findings indicate that replacement of His606 in motif II by alanine renders ATP hydrolysis less dependent on an RNA cofactor, an observation that has been described for the NPH-II and the NS3 helicases (GROSS and SHUMAN 1995 ; KIM et al. 1997 ).

These findings indicate that the ATPase activity of Prp22, although necessary, is insufficient to effect spliceosome disassembly; i.e., there is not a strict correlation between the extent to which ATP can be hydrolyzed and the protein's function in mRNA release.

RNA unwinding by Prp22 mutant proteins:
We tested helicase activity of the T4 (T637A-M853I) and T7 (T637A-G813S) suppressor proteins, using an RNA substrate with a 25-bp duplex region and 82-nucleotide and 78-nucleotide 3' tails (Fig 6). The RNA substrate (25 fmol) was incubated without protein or in the presence of wild-type Prp22 and the mutant proteins for 30 min at 37°. The labeled product of the reaction comigrated during electrophoresis with the single-stranded species released by thermal denaturation of the substrate (Fig 6, lane T). Single-stranded RNA was produced by wild-type Prp22 and 30 ng of protein unwound 50% of the dsRNA substrate (increasing the amount of protein did not yield more single-stranded product). In contrast, 30 ng of the T637A mutant unwound 5% of the substrate. The equivalent amount of the T4 (T637A-M853I) suppressor protein unwound 20%. We surmise that the M853I mutation suppresses the growth defect and the mRNA release deficiency of T637A by restoring partial RNA unwinding activity, a correlation that has been described for S27 (S635A-V539I), a suppressor mutant of S635A (SCHWER and MESZAROS 2000 ).

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 6. RNA unwinding activity. Helicase reaction mixtures containing 25 fmol of 3'-tailed RNA duplex substrate and recombinant Prp22 proteins as indicated were incubated at 37° for 30 min. The reaction products were resolved by native PAGE and the extent of unwinding was quantitated using the STORM phosphorimager.

On the other hand, the T7 (T637A-G813S) protein did not unwind the 25-bp RNA duplex at all. This suggests that the G813S mutation, which restored full spliceosome disassembly activity to T637A (Fig 4), suppressed by a mechanism that was not reflected in the assay measuring unwinding of a 25-bp duplex RNA.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study illuminates the mechanism of Prp22 function in splicing in the following ways: (1) mRNA release is a temperature-dependent process; (2) gain-of-function mutations that suppress release-defective prp22-cs mutants map to the ATPase domain; (3) mRNA release activity does not strictly correlate with the activity of Prp22 to unwind a synthetic 25-bp duplex RNA.

The ATPase activity of Prp22 is necessary but not sufficient to effect mRNA release from the spliceosome. Prp22 couples the chemical energy to a conformational step, such as to disrupt or remodel macromolecular interactions in the spliceosome, thereby leading to mRNA release. Previous studies suggested that unwinding of a 3'-tailed 25-bp duplex RNA substrate by Prp22 in vitro is a direct "read-out" of its mRNA release activity (SCHWER and MESZAROS 2000 ). This conjecture is upheld by the present analysis of the Prp22 mutant protein T4 (T637A-M853I), as well as by the analysis of the Prp22 mutants S3 (S635A-A760V), S13 (S635A-K816A), S25 (S635A-F643I), and S4 (S635A-S1058L) (B. SCHWER, unpublished data). However, the T7 (T637A-G813S) protein catalyzes mRNA release without exhibiting RNA helicase activity in vitro. It is likely that disruption of the 25-bp RNA helix used to measure RNA helicase activity in vitro requires successive cycles of ATP binding, hydrolysis, and unwinding. In contrast, the postulated RNA base-pairing interactions in the spliceosome are not very extensive and Prp22 may need only to disrupt a short duplex. This is consistent with the observed temperature dependence of mRNA release by the Prp22-T637A and Prp22-H606A mutants. It is plausible that thermal energy can compensate in vivo and in vitro for the defect in coupling ATP hydrolysis to mRNA release by weakening macromolecular contacts in the spliceosome.

The spliceosomal target for Prp22 action is unknown and it remains to be seen whether Prp22 disrupts RNA/RNA interactions or whether it functions as an "RNPase" to disrupt RNA/protein interactions in the spliceosome. Studies of vaccinia virus NPH-II, a processive 3'-to-5' RNA helicase, have demonstrated that this DExH-box protein is capable of disrupting RNA-protein interactions in addition to RNA duplexes (JANKOWSKY et al. 2001 ). RNPase activity has also been imputed to the DExD-box splicing factors Prp28 and Sub2 during spliceosome assembly on the basis of genetic studies (CHEN et al. 2001 ; KISTLER and GUTHRIE 2001 ; SCHWER 2001 ). Prp28 is involved in disruption of the U1 snRNP interaction with the 5' splice site and a role for Sub2 appears to be displacement of the Mud2 protein from the branchpoint region of the precursor RNA prior to U2 snRNP binding (CHEN et al. 2001 ; KISTLER and GUTHRIE 2001 ).

The defects of the prp22-T637A and prp22-H606A mutants can be overcome in vivo and in vitro by secondary mutations in the ATPase domain. It is possible that the intragenic mutations restore the coupling of ATP hydrolysis to mRNA release by altering the internal domain dynamics. In this regard, it is informative to relate our mutational findings for Prp22 to structural information of the DExH helicase NS3 (YAO et al. 1997 ; KIM et al. 1998 ). Although no crystal structure is available for Prp22, SANCHEZ et al. 2000 derived a structural model of Prp22 based on the NS3 structure. Their model (spanning amino acids 498–819) includes two globular domains—one of which contains motifs I and II and the other motif VI. To illustrate the interface between the globular domains, we "cracked open" the model by an 90° retroflexion of the backbone at residue 640 (Fig 7). It can be seen that the essential surface-exposed side chains of motifs I and II face those in motif VI (Fig 7A) and that motif III is located on the segment connecting the two domains (Fig 7B). This model is consistent with a ratchet-like mechanism for RNA unwinding, proposed by KIM et al. 1998 , in which a series of conformational changes that are coupled to ATP binding and hydrolysis propel translocation of the protein along the nucleic acid.

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Structural model of the Prp22 catalytic domain. The model of the catalytic domain of Prp22 from amino acids 498 to 819 derived by SANCHEZ et al. 2000 is rendered as a space-filling surface view (left) and a worm trace (right). The view is looking at the interdomain cleft, which was exposed by breaking open the structure via 90° rotation of the domain around the peptide bond at residue 640. The images were prepared using GRASP (NICHOLLS et al. 1991 ). Amino acids of Prp22 at which Ala substitutions were lethal (A) or elicited a cs growth phenotype (B) are highlighted in color and labeled on the worm trace. The positions at which second-site mutations suppressed the cs growth of H606A, T637A, and S636A are highlighted in C.

A subset of amino acids that were mutated in suppressors of T637A, H606A, and S635A are indicated in Fig 7C. It is plausible that mutations in the cleft directly affect the closing of the cleft upon ATP binding or its opening upon ATP hydrolysis. Other changes that restored growth of the mutant strains were found in amino acids that are modeled as being on the external surface of the protein and they may influence domain dynamics indirectly by affecting interactions of Prp22 with other spliceosomal components. Future studies will test the implications of this model.

Much is known about Prp22, the enzyme that catalyzes release of mature RNA from the spliceosome. However, understanding the mechanism of mRNA release in detail also requires the analysis of the macromolecular interactions within the spliceosome that are resolved or remodeled by Prp22. The analysis of extragenic suppressors of prp22-cs mutants may uncover potential spliceosomal targets (RNAs and/or proteins) for Prp22 action.

	ACKNOWLEDGMENTS

We thank Chris Lima for all his help with the structural model. The Department of Microbiology and Immunology acknowledges the support of the William Randolph Hearst Foundation. This work was supported by grant GM50288 from the National Institutes of Health.

Manuscript received July 31, 2001; Accepted for publication November 5, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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