Structure-guided mutational analysis of a yeast DEAD-box protein involved in mitochondrial RNA splicing

Abstract

DEAD-box proteins are RNA-dependent ATPase enzymes that have been implicated in nearly all aspects of RNA metabolism. Since many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acid, and to promote helix unwinding, DEAD-box proteins are generally thought to modulate RNA structure in vivo. However, the extent to which these enzymatic properties are important for the in vivo functions of DEAD-box proteins remains unclear. To evaluate how these properties influence DEAD-box protein native function, we probed the importance of several highly conserved residues in the yeast DEAD-box protein Mss116p, which is required for the splicing of all mitochondrial catalytic introns in Saccharomyces cerevisiae. Using an MSS116 deletion strain, we have expressed plasmid-borne variants of MSS116 containing substitutions in residues predicted to be important for extensive networks of interactions required for ATP hydrolysis and helix unwinding. We have analyzed the importance of these residues to the splicing functions of Mss116p in vivo and compared these results with the biochemical properties of recombinant proteins determined here and in previously published work. We observed that the efficiency by which an Mss116p variant catalyzes ATP hydrolysis correlates with facilitating mitochondrial splicing, while efficient helix unwinding appears to be insufficient for splicing. In addition, we show that each splicing-defective variant affects the splicing of structurally diverse introns to the same degree. Together, these observations suggest that the efficiency by which Mss116p catalyzes the hydrolysis of ATP is critical for all of its splicing functions in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, these observations support a model in which enzyme turnover is a crucial factor in Mss116p splicing function. These results are discussed in the context of current models of Mss116p-facilitated splicing.

Fig. 1

Networks of conserved interdomain interactions in “closed” conformation of Mss116p and additional DEAD-box proteins. (a) Schematic of Mss116p conserved sequence motifs. Motifs Q through III are located in the N-terminal domain, and motifs IV through VI are located in the C-terminal domain. The motifs that form interdomain interactions are colored, and the dotted lines represent interactions between motifs.; ; ; ; ; ; Residues in “bold” are the specific residues mutated in this investigation (K158A, R245E, S305A, T307A, Q412A). (b) Structural representations of interactions involving motifs III and QxxR. The green dotted lines represent hydrogen bonds between residues. The crystal structure of the Mss116p protein bound to a polyU-RNA and AMP-PNP was used to create these figures (see Materials and Methods). These interactions have been predicted from additional DEAD-box protein crystal structures.; ; ; ; ; ; For clarity the interaction between the backbone of T307 and the side chain of S305 is not shown, as well as multiple interactions involving the backbone of Q412.

Fig. 2

Growth phenotypes of wild-type and mutant derivatives of Mss116p. CEN6 plasmids containing wild-type (W.T.) and mutant derivatives of Mss116p (K158A, T307A, Q412A) or the pRS415 vector control (E.V.) were transformed into the wild-type (w.t.) or mss116Δ strains. Cells were grown in liquid, dextrose-containing medium at 30 °C, serially diluted, spotted on dextrose- or glycerol-containing medium, and incubated at the indicated temperatures.

Fig. 3

Growth phenotypes of wild-type and mutant derivatives of Mss116p. CEN6 plasmids containing wild-type (W.T.) and mutant derivatives of Mss116p (S305A, T307A) or the pRS415 vector control (E.V.) were transformed into the wild-type (w.t.) or mss116Δ strains. Cells were grown in liquid, dextrose-containing medium at 30 °C, serially diluted, spotted on dextrose- or glycerol-containing medium, and incubated at the indicated temperatures.

Fig. 4

Splicing phenotypes of mss116Δ strains expressing variants of Mss116p assessed by Northern blot analysis. (a) Schematic of the COX1 pre-mRNA transcript containing eight exons and seven introns, with nomenclature designated as in Valencik et. al., 1989. G.I, group I introns; G.II, group II introns. (b) Representative northern blots of total mitochondrial RNA from mss116Δ strains harboring mutant derivatives of Mss116p. RNA was harvested from cells grown in dextrose-containing media at 30 °C. Blots were hybridized with 5′-³²P-labeled DNA oligonucleotides complementary to the COX1 exons aE5γ and aE6, or the intron-less COX3 transcript, which served as the loading control. Different splicing isoforms are indicated by letters A-F. Fully processed pre-mRNAs are marked as LE for ligated exons. (c) Histogram of exon ligation in each mutant strain relative to exon ligation in the wild-type strain, as detected using both the aE5γ and aE6 exon probes. All data was normalized to the abundance of the COX3 RNA. The data using the aE5γ probe are the average from two independent experiments and the error bars represent the range of the values divided by two. Exon ligation in the strains expressing the E.V., K158A, and T307A variants was very low in abundance or essentially zero and may not be seen on the histogram.

Fig. 5

Splicing phenotypes of mss116Δ strains expressing variants of Mss116p using RT-PCR. (a) Simplified schematic of the COX1 and COB pre-mRNA transcripts. Primers used to amplify the products of aI5β, aI5γ, bI1, and bI3 splicing are indicated by arrows. (b) Representative gel images of the products of aI5β, aI5γ, bI1, and bI3 splicing after PCR cycle 26. Ligated exons were not amplified in the negative control, in which reverse transcriptase was not included in the cDNA synthesis reaction (-RT). (c) Histograms of the amount of ligated exons in each mutant strain relative to the wild-type strain. The abundance of ligated exons was normalized to the amplification of the COX3 transcript. Data is the average of two independent experiments, and the error bars represent the range of the values divided by two. Exon ligation in the strain expressing the vector control alone (E.V.) was very low in abundance or essentially zero and may not be seen on the histogram.

Fig. 6

The R245E Mss116p variant does not rescue the growth or splicing phenotypes of the mss116Δ strain. (a) Structural representation of the contacts formed by the Mss116p R245 residue, as predicted by several DEAD-box protein crystal structures (see legend of Fig. 1). Interactions formed by the Q412 residue are shown for reference. (b) CEN6 plasmids containing wild-type (W.T.) and mutant derivatives of Mss116p (K158A, R245E) or the pRS415 vector control (E.V.) were transformed into the mss116Δ strains, as described in Figs. 2 and 3. (c) Representative gel images and (d) histograms for intron splicing in each mutant strain, as described in Fig. 5.

Fig. 7

ATPase activity of wild-type and mutant derivatives of Mss116p. Representative plots of free ADP versus time for reactions with 50 μM ATP. The curves are linear fits to the data with the slopes equal to the initial velocity (v_o). For kinetic parameters, see Table 1. W.T., wild-type Mss116p; None, no protein added.

Fig. 8

Unwinding activity of wild-type and mutant derivatives of Mss116p. (a) Representative gel images for unwinding reactions in the presence and absence of ATP at 30 °C. The mobility of the released ³²P-DNA oligonucleotide was confirmed by boiling the complex and running that material alongside the starting material (not shown). (b) Representative time course plots for unwinding. Reactions were initiated by the addition of wild-type or mutant protein (1.5 μM final concentration) or protein dilution buffer (None). Aliquots were quenched at time points over a period of 45 min. Data for wild-type Mss116p (W.T.) and the S305A, T307A, and Q412A variants in the presence of ATP was fit to a first-order, double exponential equation: Fraction Duplex = A(e^-kt) + B(e^-kt) where A and B are the amplitude of duplex in each phase and k represents the first order rate constants for each phase. Data for W.T. Mss116p in the absence of ATP, R245E in the presence of ATP, and in reactions without protein was fit to a single exponential: Fraction Duplex = A(e^-kt) where A is the amplitude and k represents the first order rate constant. See Table 2 for values derived from multiple experiments and calculated errors. Unwinding by S305A in the absence of ATP was similar to the rate and amplitude of unwinding by W.T. in the absence of ATP (not shown). Multiple bands in the duplex are due to the addition of non-templated nucleotides to the RNA transcript.

Fig. 9

The importance of ATP hydrolysis within the context of the proposed models for Mss116p facilitated intron splicing. (a) In this model, Mss116p may act as an ATP-dependent RNA chaperone to unwind short, non-native helices in structured RNAs. Coupled ATP binding and non-specific RNA binding by Mss116p promotes local strand separation. ATP hydrolysis promotes enzyme turnover and the release of a destabilized RNA. The base pairs of the destabilized structure may re-anneal to re-form the non-native structure or completely dissociate and promote RNA folding to a native structure. Inefficient ATP hydrolysis and enzyme turnover could potentially diminish the relative concentration of destabilized RNAs that re-fold to a functional structure, thereby reducing the efficiency of Mss116p-facilited RNA processing. (b) A second model is based on the suggestion that Mss116p may capture a short lived, single-stranded region of a particular RNA folding intermediate through ATP-dependent RNA binding and promote the ordered folding of the remaining domains of a group II intron RNA.; In theory, this intermediate could follow folding pathways that either lead to a native structure (adjacent, parallel helices) or a non-native structure (anti-parallel helices). ATP hydrolysis and RNA release at the appropriate time within the folding pathway may increase the likelihood that the RNA will form the native, functional structure. If ATP hydrolysis and enzyme turnover are slow, the protein could sterically impede native folding. Consequently, the susceptibility of the RNA to mis-fold into a non-native structure may increase, thus reducing the efficiency of Mss116p-facilitated intron splicing.