Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity

The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.     

A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing

The Neurospora crassa CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns by inducing formation of the catalytically active RNA structure. Here, we identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing in vivo and vitro. CYT-19 does not bind specifically to group I intron RNAs and instead functions as an ATP-dependent RNA chaperone to destabilize nonnative RNA structures that constitute kinetic traps in the CYT-18-assisted RNA-folding pathway. Our results demonstrate that a DExH/D-box protein has a specific, physiologically relevant chaperone function in the folding of a natural RNA substrate.     

The Azoarcus group I intron ribozyme misfolds and is accelerated for refolding by ATP-dependent RNA chaperone proteins

Structured RNAs traverse complex energy landscapes that include valleys representing misfolded intermediates. In Neurospora crassa and Saccharomyces cerevisiae, efficient splicing of mitochondrial group I and II introns requires the DEAD box proteins CYT-19 and Mss116p, respectively, which promote folding transitions and function as general RNA chaperones. To test the generality of RNA misfolding and the activities of DEAD box proteins in vitro, here we measure native folding of a small group I intron ribozyme from the bacterium Azoarcus by monitoring its catalytic activity. To develop this assay, we first measure cleavage of an oligonucleotide substrate by the prefolded ribozyme. Substrate cleavage is rate-limited by binding and is readily reversible, with an internal equilibrium near unity, such that the amount of product observed is less than the amount of native ribozyme. We use this assay to show that approximately half of the ribozyme folds readily to the native state, whereas the other half forms an intermediate that transitions slowly to the native state. This folding transition is accelerated by urea and increased temperature and slowed by increased Mg(2+) concentration, suggesting that the intermediate is misfolded and must undergo transient unfolding during refolding to the native state. CYT-19 and Mss116p accelerate refolding in an ATP-dependent manner, presumably by disrupting structure in the intermediate. These results highlight the tendency of RNAs to misfold, underscore the roles of CYT-19 and Mss116p as general RNA chaperones, and identify a refolding transition for further dissection of the roles of DEAD box proteins in RNA folding.     

The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.     

Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E^ΔP5abc). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E^ΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.     

Unwinding by Local Strand Separation Is Critical for the Function of DEAD-Box Proteins as RNA Chaperones

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.     

RNA chaperone activity and RNA-binding properties of the E. coli protein StpA

The E. coli protein StpA has RNA annealing and strand displacement activities and it promotes folding of RNAs by loosening their structures. To understand the mode of action of StpA, we analysed the relationship of its RNA chaperone activity to its RNA-binding properties. For acceleration of annealing of two short RNAs, StpA binds both molecules simultaneously, showing that annealing is promoted by crowding. StpA binds weakly to RNA with a preference for unstructured molecules. Binding of StpA to RNA is strongly dependent on the ionic strength, suggesting that the interactions are mainly electrostatic. A mutant variant of the protein, with a glycine to valine change in the nucleic-acid-binding domain, displays weaker RNA binding but higher RNA chaperone activity. This suggests that the RNA chaperone activity of StpA results from weak and transient interactions rather than from tight binding to RNA. We further discuss the role that structural disorder in proteins may play in chaperoning RNA folding, using bioinformatic sequence analysis tools, and provide evidence for the importance of conformational disorder and local structural preformation of chaperone nucleic-acid-binding sites.     

DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome.     

Design and development of a catalytic ribonucleoprotein

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.     

Slow folding kinetics of RNase P RNA.

Understanding the folding mechanisms of large, highly structured RNAs is important for understanding how these molecules carry out their function. Although models for the three-dimensional architecture of several large RNAs have been constructed, the process by which these structures are formed is only now beginning to be explored. The kinetic folding pathway of the Tetrahymena ribozyme involves multiple intermediates and both Mg2+-dependent and Mg2+-independent steps. To determine whether this general mechanism is representative of folding of other large RNAs, a study of RNase P RNA folding was undertaken. We show, using a kinetic oligonucleotide hybridization assay, that there is at least one slow step on the folding pathway of RNase P RNA, resulting in conformational changes in the P7 helix region on the minute timescale. Although this folding event requires the presence of Mg2+, the slow step itself does not involve Mg2+ binding. The P7 and P2 helix regions exhibit distinctly different folding behavior and ion dependence, implying that RNase P folding is likely to be a complex process. Furthermore, there are distinct similarities in the folding of RNase P RNA from both Bacillus subtilis and Escherichia coli, indicating that the folding pathway may also be conserved along with the final structure. The slow folding kinetics, Mg2+-independence of the rate, and existence of intermediates are basic features of the folding mechanism of the Tetrahymena group I intron that are also found in RNase P RNA, suggesting these may be general features of the folding of large RNAs.     

A phage RNA-binding protein binds to a non-cognate structured RNA and stabilizes its core structure

Recent studies suggest that some RNA-binding proteins facilitate the folding of non-cognate RNAs. Here, we report that bacteriophage MS2 coat protein (MS2 CP) bound and promoted the catalytic activity of Candida group I ribozyme. Cloning of the MS2-bound RNA segments showed that this protein primarily interacts with the P5ab-P5 structure. Ultraviolet cross-linking and the T1 footprinting assay further showed that MS2 binding stabilized tertiary interactions, including the conserved L9-P5 interaction, and led to a more compact core structure. This mechanism is similar to that of the yeast mitochondrial tyrosyl-tRNA synthetase on other group I introns, suggesting that different RNA-binding proteins may use common mechanisms to support RNA structures.     

Protein-dependent transition states for ribonucleoprotein assembly

Native folding and splicing by the Saccharomyces cerevisiae mitochondrial bI5 group I intron RNA is facilitated by both the S. cerevisiae CBP2 and Neurospora crassa CYT-18 protein cofactors. Both protein-bI5 RNA complexes splice at similar rates, suggesting that the RNA active site structure is similar in both ribonucleoproteins. In contrast, the two proteins assemble with the bI5 RNA by distinct mechanisms and bind opposing, but partially overlapping, sides of the group I intron catalytic core. Assembly with CBP2 is limited by a slow, unimolecular RNA folding step characterized by a negligible activation enthalpy. We show that assembly with CYT-18 shows four distinctive features. (1) CYT-18 binds stably to the bI5 RNA at the diffusion controlled limit, but assembly to a catalytically active RNA structure is still limited by RNA folding, as visualized directly using time-resolved footprinting. (2) This mechanism of rapid stable protein binding followed by subsequent assembly steps has a distinctive kinetic signature: the apparent ratio of k(off) to k(on), determined in a partitioning experiment, differs from the equilibrium K(d) by a large factor. (3) Assembly with CYT-18 is characterized by a large activation enthalpy, consistent with a rate limiting conformational rearrangement. (4) Because assembly from the kinetically trapped state is faster at elevated temperature, we can identify conditions where CYT-18 accelerates (catalyzes) bI5 RNA folding relative to assembly with CBP2.     

Analysis of the CYT-18 Protein Binding Site at the Junction of Stacked Helices in a Group I Intron RNA by Quantitative Binding Assays andin vitroSelection

TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. Previous studies showed that CYT-18 binds with high affinity to the P4-P6 domain of the catalytic core and that there is some additional contribution to binding from the P3-P9 domain. Here, quantitative binding assays with deletion derivatives of theN. crassamitochondrial large rRNA intron showed that at least 70% of the binding energy can be accounted for by the interaction of CYT-18 with the P4-P6 domain. Within this domain, P4 and P6 are required for high affinity CYT-18 binding, while the distal elements P5 and P6a may contribute indirectly by stabilizing the correct structure of the binding site in P4 and P6. CYT-18 binds to a small RNA corresponding to the isolated P4-P6 domain, but not to a permuted version of this RNA in which P4-P6 is a continuous rather than a stacked helix. Iterativein vitroselection experiments with the isolated P4-P6 domain showed a requirement for base-pairing to maintain helices P4, P6 and P6a, but indicate that P5 is subject to fewer constraints. The most strongly conserved nucleotides in the selections were clustered around the junction of the P4-P6 stacked helix, with ten nucleotides (J3/4-2,3, P4 bp -1 and 3, and P6 bp -1 and 2) found invariant in the context of the wild-type RNA structure.In vitromutagenesis confirmed that replacement of the wild-type nucleotides at J3/4-2 and 3 or P4 bp-3 markedly decreased CYT-18 binding, reflecting either base specific contacts or indirect readout of RNA structure by the protein. Our results suggest that a major function of CYT-18 is to promote assembly of the P4-P6 domain by stabilizing the correct geometry at the junction of the P4-P6 stacked helix. The relatively large number of conserved nucleotides at the binding site suggests that the interaction of CYT-18 with group I introns is unlikely to have arisen by chance and could reflect either an evolutionary relationship between group I introns and tRNAs or interaction with a common stacked-helical structural motif that evolved separately in these RNAs.     

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.     

Two major tertiary folding transitions of the Tetrahymena catalytic RNA.

The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.     

Characterizing the structural features of RNA/RNA interactions of the F-plasmid FinOP fertility inhibition system

F-like plasmid transfer is mediated by the FinOP fertility inhibition system. Expression of the F positive regulatory protein, TraJ, is controlled by the action of the antisense RNA, FinP, and the RNA-binding protein FinO. FinO binds to and protects FinP from degradation and promotes duplex formation between FinP and traJ mRNA, leading to repression of both traJ expression and conjugative F transfer. FinP antisense RNA secondary structure is composed of two stem-loops separated by a 4-base single-stranded spacer and flanked on each side by single-stranded tails. Here we show that disruption of the expected Watson-Crick base pairing between the loops of FinP stem-loop I and its cognate RNA binding partner, traJ mRNA stem-loop Ic, led to a moderate reduction in the rate of duplex formation in vitro. In vivo, alterations of the anti-ribosome binding site region in the loop of FinP stem-loop I reduced the ability of the mutant FinP to mediate fertility inhibition and to inhibit TraJ expression when expressed in trans at an elevated copy number. Alterations of intermolecular complementarity between the stems of these RNAs reduced the rate of duplex formation. Our results suggest that successful interaction between stem-loop I of FinP and stem-loop Ic of traJ mRNA requires that base pairing must proceed from an initial loop-loop interaction through the top portion of the stems for stable duplex formation to occur.     

ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo

The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ,? we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.     

Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La''s RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.     

Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein

We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli. Huang et al. The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs. To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein. RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed. Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA. More interestingly, the C-terminal domain was a target of protein kinase C (PKC). Phosphorylation of the C-terminal domain of p68 abolished its RNA binding. Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68. The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein.     

A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface

We determined a 1.95 A X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18's nucleotide binding fold and intermediate alpha-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNATyr. Our results show how essential proteins can progressively evolve new functions.