A recurrent RNA-binding domain is appended to eukaryotic aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases of higher eukaryotes possess polypeptide extensions in contrast to their prokaryotic counterparts. These extra domains of poorly understood function are believed to be involved in protein-protein or protein-RNA interactions. Here we showed by gel retardation and filter binding experiments that the repeated units that build the linker region of the bifunctional glutamyl-prolyl-tRNA synthetase had a general RNA-binding capacity. The solution structure of one of these repeated motifs was also solved by NMR spectroscopy. One repeat is built around an antiparallel coiled-coil. Strikingly, the conserved lysine and arginine residues form a basic patch on one side of the structure, presenting a suitable docking surface for nucleic acids. Therefore, this repeated motif may represent a novel type of general RNA-binding domain appended to eukaryotic aminoacyl-tRNA synthetases to serve as a cis-acting tRNA-binding cofactor.     

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.     

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.     

Functional redundancy in the nonspecific RNA binding domain of a class I tRNA synthetase

The sequence of a 228-amino acid nonspecific RNA binding domain appended to the N terminus of a eukaryote tRNA synthetase is shown here to have two lysine-rich clusters (LRCs) that are functionally significant in vivo and in vitro. These two LRCs have unrelated sequences and are separated by a spacer of over 100 amino acids. By using a sensitive test for function in vivo, each LRC is shown to be sufficient in the absence of the other. This sufficiency requires fusion of the spacer to either of the LRCs. Experiments in vitro confirmed that the LRCs are each important for RNA binding. Thus, this nonspecific RNA binding domain has two dissimilar lysine-rich sequence elements that are functionally redundant. Further experiments suggest that this redundancy is not used to dock two molecules of RNA but rather to enhance the overall affinity for a single RNA molecule.     

Fibulin-1 acts as a cofactor for the matrix metalloprotease ADAMTS-1

ADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression.     

A minihelix-loop RNA acts as a trans-aminoacylation catalyst.

We previously reported a bifunctional ribozyme that catalyzes self-aminoacylation and subsequent acyl-transfer to a tRNA. The ribozyme selectively recognizes a biotinyl-glutamine substrate, and charges the tRNA molecule in trans. Structurally, there are two catalytic domains, referred to as glutamine-recognition (QR) and acyl-transferase (ATRib). We report here the essential catalytic core of the QR domain as determined by extensive biochemical probing, mutation, and structural minimization. The minimal core of the QR domain is a 29-nt helix-loop RNA, which is also able to glutaminylate ATRib in trans. Its amino acid binding site is embedded in an 11-nt cluster that is adjacent to the loop that interacts with the ATRib domain. Our study shows that a minihelix-loop RNA can act as a trans-aminoacylation catalyst, which lends support for the critical role of minihelix-loops in the early evolution of the aminoacylation system.     

Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.     

A substrate-assisted concerted mechanism for aminoacylation by a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.     

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein''s asymmetry, its large occluded site size, and its RNA folding activity.     

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.     

Requirement for C-terminal extension to the RNA binding domain for efficient RNA binding by ribosomal protein L2

Ribosomal protein L2 is a primary 23S rRNA binding protein in the large ribosomal subunit. We examined the contribution of the N- and C-terminal regions of Bacillus stearothermophilus L2 (BstL2) to the 23S rRNA binding activity. The mutant desN, in which the N-terminal 59 residues of BstL2 were deleted, bound to the 23S rRNA fragment to the same extent as wild type BstL2, but the mutation desC, in which the C-terminal 74 amino acid residues were deleted, abolished the binding activity. These observations indicated that the C-terminal region is involved in 23S rRNA binding. Subsequent deletion analysis of the C-terminal region found that the C-terminal 70 amino acids are required for efficient 23S rRNA binding by BstL2. Furthermore, the surface plasmon resonance analysis indicated that successive truncations of the C-terminal residues increased the dissociation rate constants, while they had little influence on association rate constants. The result indicated that reduced affinities of the C-terminal deletion mutants were due only to higher dissociation rate constants, suggesting that the C-terminal region primarily functions by stabilizing the protein L2-23S rRNA complex.     

The light chain binding domain of expressed smooth muscle heavy meromyosin acts as a mechanical lever

Structural data led to the proposal that the molecular motor myosin moves actin by a swinging of the light chain binding domain, or "neck." To test the hypothesis that the neck functions as a mechanical lever, smooth muscle heavy meromyosin (HMM) mutants were expressed with shorter or longer necks by either deleting or adding light chain binding sites. The mutant HMMs were characterized kinetically and mechanically, with emphasis on measurements of unitary displacements and forces in the laser trap assay. Two shorter necked constructs had smaller unitary step sizes and moved actin more slowly than WT HMM in the motility assay. A longer necked construct that contained an additional essential light chain binding site exhibited a 1.4-fold increase in the unitary step size compared with its control. Kinetic changes were also observed with several of the constructs. The mutant lacking a neck produced force at a somewhat reduced level, while the force exerted by the giraffe construct was higher than control. The single molecule displacement and force data support the hypothesis that the neck functions as a rigid lever, with the fulcrum for movement and force located at a point within the motor domain.     

cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

The structural requirements of the hydrophobic domain contained in poliovirus polypeptide 3AB were studied by using a molecular genetic approach in combination with an in vitro biochemical analysis. We report here the generation and analysis of deletion, insertion, and amino acid replacement mutations aimed at decreasing the hydrophobic character of the domain. Our results indicated that the hydrophobicity of this region of 3AB is necessary to maintain normal viral RNA synthesis. However, in vitro membrane association assays of the mutated proteins did not establish a direct correlation between 3AB membrane association and viral RNA synthesis. Some of the lethal mutations we engineered produced polyproteins with abnormal P2- and P3-processing capabilities due to an alteration in the normal cleavage order of the polyprotein. A detailed analysis of these mutants suggests that P2 is not the major precursor for polypeptides 2A and 2BC and that P2 protein products are derived from P2-P3-containing precursors (most likely P2-P3 or P2-3AB). Such precursors are likely to result from primary polyprotein cleavage events that initiate a proteolytic cascade not previously documented. Our results also indicated that the function provided by the hydrophobic domain of 3AB cannot be provided in trans. We discuss the implications of these results on the formation of limited-diffusion replication complexes as a means of sequestering P2- and P3-region polypeptides required for RNA synthesis and protein processing.     

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule-dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA-binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.     

An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme

RNA editing by adenosine deamination is particularly prevalent in the squid nervous system. We hypothesized that the squid editing enzyme might contain structural differences that help explain this phenomenon. As a first step, a squid adenosine deaminase that acts on RNA (sqADAR2a) cDNA and the gene that encodes it were cloned from the giant axon system. PCR and RNase protection assays showed that a splice variant of this clone (sqADAR2b) was also expressed in this tissue. Both versions are homologous to the vertebrate ADAR2 family. sqADAR2b encodes a conventional ADAR2 family member with an evolutionarily conserved deaminase domain and two double-stranded RNA binding domains (dsRBD). sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an “extra” dsRBD. Both splice variants are expressed at comparable levels and are extensively edited, each in a unique pattern. Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. Using a standard 48-h protein induction, both sqADAR2a and sqADAR2b exhibit promiscuous self-editing; however, this activity is particularly robust for sqADAR2a. By decreasing the induction time to 16 h, self-editing was mostly eliminated. We next tested the ability of sqADAR2a and sqADAR2b to edit two K⁺ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. These data suggest that the “extra” dsRBD confers high activity on sqADAR2a.     

A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection

Cyclophilins are peptidyl prolyl cis-trans isomerases that are highly conserved throughout eukaryotes and that are best known for being the cellular target of the immunosuppressive drug cyclosporin A (CsA). The activity of CsA is caused by the drug forming a complex with cyclophilin A and inhibiting the calmodulin-dependent phosphoprotein phosphatase calcineurin. We have investigated the role of CYP1, a cyclophilin-encoding gene in the phytopathogenic fungus Magnaporthe grisea, which is the causal agent of rice blast disease. CYP1 putatively encodes a mitochondrial and cytosolic form of cyclophilin, and targeted gene replacement has shown that CYP1 acts as a virulence determinant in rice blast. Cyp1 mutants show reduced virulence and are impaired in associated functions, such as penetration peg formation and appressorium turgor generation. CYP1 cyclophilin also is the cellular target for CsA in Magnaporthe, and CsA was found to inhibit appressorium development and hyphal growth in a CYP1-dependent manner. These data implicate cyclophilins as virulence factors in phytopathogenic fungi and also provide evidence that calcineurin signaling is required for infection structure formation by Magnaporthe.     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

A unique glutamic acid-lysine (EK) domain acts as a splicing inhibitor

SRrp86 is a unique member of the SR protein superfamily of splicing factors containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK) rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins as long as the entire region encompassing the RS-EK-RS domains was intact. To further investigate the function and domains of SRrp86, we generated a series of chimeric proteins by swapping the RNA recognition motif and RS domains between SRrp86 and two canonical members of the SR superfamily, ASF/SF2 and SRp75. Although domain swaps between SRrp86 and ASF/SF2 showed that the RRMs primarily determined splicing activity, swaps between SRrp86 and SRp75 demonstrated that the RS domains could also determine activity. Because SRp75 also has two RS domains but lacks the EK domain, we further investigated the role of the EK domain and found that it acts to repress splicing and splice-site selection, both in vitro and in vivo. Incubation of extracts with peptides encompassing the EK-rich region inactivated splicing and insertion of the EK region into SRp75 abolished its ability to activate splicing. Thus, the unique EK domain of SRrp86 plays a modulatory role controlling RS domain function.