30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC.

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine analog to trap the complex in a postcatalytic state to enable determination of a global 3.0 Å cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder-to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to resensitize bacterial pathogens to aminoglycosides.

Role of helical structure and dynamics in oligoadenylate synthetase 1 (OAS1) mismatch tolerance and activation by short dsRNAs.

The 2'-5'-oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) that play a critical role in limiting viral infection. How these proteins are able to avoid aberrant activation by cellular RNAs is not fully understood, but adenosine-to-inosine (A-to-I) editing has been proposed to limit accumulation of endogenous RNAs that might otherwise cause stimulation of the OAS/RNase L pathway. Here, we aim to uncover whether and how such sequence modifications can restrict the ability of short, defined dsRNAs to activate the single-domain form of OAS, OAS1. Unexpectedly, we find that all tested inosine-containing dsRNAs have an increased capacity to activate OAS1, whether in a destabilizing (I•U) or standard Watson-Crick-like base pairing (I-C) context. Additional variants with strongly destabilizing A•C mismatches or stabilizing G-C pairs also exhibit increased capacity to activate OAS1, eliminating helical stability as a factor in the relative ability of the dsRNAs to activate OAS1. Using thermal difference spectra and molecular dynamics simulations, we identify both increased helical dynamics and specific local changes in helical structure as important factors in the capacity of short dsRNAs to activate OAS1. These helical features may facilitate more ready adoption of the distorted OAS1-bound conformation or stabilize important structures to predispose the dsRNA for optimal binding and activation of OAS1. These studies thus reveal the molecular basis for the greater capacity of some short dsRNAs to activate OAS1 in a sequence-independent manner.

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.

Changes in bacterial ribosomal RNA (rRNA) methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2'-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a postcatalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144, which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes, even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Ensuring a sustained workforce to combat antibiotic resistance in the 21st century: the critical need for training the next-gen of scientists at the pre-doctoral level.

Antibiotics revolutionized the treatment of bacterial infection and enhanced modern healthcare. While the current global antibiotic resistance crisis is widely acknowledged, the need for a highly trained cohort of scientists to continue and expand efforts to combat this threat has received less attention. We posit that training of pre-doctoral students in the antibiotic resistance field is critical for future efforts in combating this crisis and urge development of training programs that focus on this need.

Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases.

The SpoU-TrmD (SPOUT) methyltransferase superfamily was designated when structural similarity was identified between the transfer RNA-modifying enzymes TrmH (SpoU) and TrmD. SPOUT methyltransferases are found in all domains of life and predominantly modify transfer RNA or ribosomal RNA substrates, though one instance of an enzyme with a protein substrate has been reported. Modifications placed by SPOUT methyltransferases play diverse roles in regulating cellular processes such as ensuring translational fidelity, altering RNA stability, and conferring bacterial resistance to antibiotics. This large collection of S-adenosyl-L-methionine-dependent methyltransferases is defined by a unique α/ß fold with a deep trefoil knot in their catalytic (SPOUT) domain. Herein, we describe current knowledge of SPOUT enzyme structure, domain architecture, and key elements of catalytic function, including S-adenosyl-L-methionine co-substrate binding, beginning with a new sequence alignment that divides the SPOUT methyltransferase superfamily into four major clades. Finally, a major focus of this review will be on our growing understanding of how these diverse enzymes accomplish the molecular feat of specific substrate recognition and modification, as highlighted by recent advances in our knowledge of protein-RNA complex structures and the discovery of the dependence of one SPOUT methyltransferase on metal ion binding for catalysis. Considering the broad biological roles of RNA modifications, developing a deeper understanding of the process of substrate recognition by the SPOUT enzymes will be critical for defining many facets of fundamental RNA biology with implications for human disease.

Human OAS1 activation is highly dependent on both RNA sequence and context of activating RNA motifs.

2'-5'-Oligoadenylate synthetases (OAS) are innate immune sensors of cytosolic double-stranded RNA (dsRNA) and play a critical role in limiting viral infection. dsRNA binding induces allosteric structural changes in OAS1 that reorganize its catalytic center to promote synthesis of 2'-5'-oligoadenylate and thus activation of endoribonuclease L. Specific RNA sequences and structural motifs can also enhance activation of OAS1 through currently undefined mechanisms. To better understand these drivers of OAS activation, we tested the impact of defined sequence changes within a short dsRNA that strongly activates OAS1. Both in vitro and in human A549 cells, appending a 3'-end single-stranded pyrimidine (3'-ssPy) can strongly enhance OAS1 activation or have no effect depending on its location, suggesting that other dsRNA features are necessary for correct presentation of the motif to OAS1. Consistent with this idea, we also find that the dsRNA binding position is dictated by an established consensus sequence (WWN9WG). Unexpectedly, however, not all sequences fitting this consensus activate OAS1 equivalently, with strong dependence on the identity of both partially conserved (W) and non-conserved (N9) residues. A picture thus emerges in which both specific RNA features and the context in which they are presented dictate the ability of short dsRNAs to activate OAS1.

Dysregulation of M segment gene expression contributes to influenza A virus host restriction.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.

Targeted Redesign of Suramin Analogs for Novel Antimicrobial Lead Development.

The emergence of new viral infections and drug-resistant bacteria urgently necessitates expedient therapeutic development. Repurposing and redesign of existing drugs against different targets are one potential way in which to accelerate this process. Suramin was initially developed as a successful antiparasitic drug but has also shown promising antiviral and antibacterial activities. However, due to its high conformational flexibility and negative charge, suramin is considered quite promiscuous toward positively charged sites within nucleic acid binding proteins. Although some suramin analogs have been developed against specific targets, only limited structure-activity relationship studies were performed, and virtual screening has yet to be used to identify more specific inhibitor(s) based on its scaffold. Using available structures, we investigated suramin's target diversity, confirming that suramin preferentially binds to protein pockets that are both positively charged and enriched in aromatic or leucine residues. Further, suramin's high conformational flexibility allows adaptation to structurally diverse binding surfaces. From this platform, we developed a framework for structure- and docking-guided elaboration of suramin analog scaffolds using virtual screening of suramin and heparin analogs against a panel of diverse therapeutically relevant viral and bacterial protein targets. Use of this new framework to design potentially specific suramin analogs is exemplified using the SARS-CoV-2 RNA-dependent RNA polymerase and nucleocapsid protein, identifying leads that might inhibit a wide range of coronaviruses. The approach presented here establishes a computational framework for designing suramin analogs against different bacterial and viral targets and repurposing existing drugs for more specific inhibitory activity.

Substrate recognition by the Pseudomonas aeruginosa EF-Tu-modifying methyltransferase EftM.

The opportunistic bacterial pathogen Pseudomonas aeruginosa is a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, or severe burns. P. aeruginosa adhesion to host epithelial cells is enhanced by surface-exposed translation elongation factor EF-Tu carrying a Lys-5 trimethylation, incorporated by the methyltransferase EftM. Thus, the EF-Tu modification by EftM may represent a target to prevent P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM activity by defining the molecular mechanism by which it recognizes EF-Tu. Acting on the observation that EftM can bind to EF-Tu lacking its N-terminal peptide (encompassing the Lys-5 target site), we generated an EftM homology model and used it in protein/protein docking studies to predict EftM/EF-Tu interactions. Using site-directed mutagenesis of residues in both proteins, coupled with binding and methyltransferase activity assays, we experimentally validated the predicted protein/protein interface. We also show that EftM cannot methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to enable placement of the first 5-6 amino acids of EF-Tu into a conserved peptide-binding channel in EftM. In this channel, a group of residues that are highly conserved in EftM proteins position the N-terminal sequence to facilitate Lys-5 modification. Our findings reveal that EftM employs molecular strategies for substrate recognition common among both class I (Rossmann fold) and class II (SET domain) methyltransferases and pave the way for studies seeking a deeper understanding of EftM's mechanism of action on EF-Tu.

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition.

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase-substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

Antibiotic Substrate Selectivity of Pseudomonas aeruginosa MexY and MexB Efflux Systems Is Determined by a Goldilocks Affinity.

Resistance-nodulation-division (RND) efflux pumps are important contributors to bacterial antibiotic resistance. In this study, we combined evolutionary sequence analyses, computational structural modeling, and ligand docking to develop a framework that can explain the known antibiotic substrate selectivity differences between two Pseudomonas aeruginosa RND transporters, MexY and MexB. For efficient efflux, antibiotic substrates must possess a "Goldilocks affinity": binding strong enough to allow interaction with transporter but not so tight as to impede movement through the pump.

A human cellular noncoding RNA activates the antiviral protein 2'-5'-oligoadenylate synthetase 1.

The 2'-5'-oligoadenylate synthetase (OAS) family of enzymes sense cytosolic dsRNA, a potent signal of viral infection. In response to dsRNA binding, OAS proteins synthesize the second messenger 2'-5'-linked oligoadenylate that activates the latent ribonuclease L (RNase L). RNase L-mediated degradation of viral and cellular RNAs effectively halts viral replication and further stimulates innate immune responses by inducing type I interferon. The OAS/RNase L pathway is therefore central in innate immune recognition and promotion of antiviral host responses. However, the potential for specific RNA sequences or structures to drive OAS1 activation and the molecular mechanisms by which they act are not currently fully understood. Moreover, the cellular regulators of OAS activity are not well defined. Here, we demonstrate that the human cellular noncoding RNA 886 (nc886) activates OAS1 both in vitro and in human A549 cells. We show that a unique structure present only in one of the two structural conformers adopted by nc886 drives potent OAS1 activation. In contrast, the conformer lacking this unique structure activated OAS1 only very weakly. We also found that formation of this OAS1-activating structural motif depends on the nucleotides in the apical-most loop of nc886 and the adjacent helix. These findings identify a cellular RNA capable of activating the OAS/RNase L pathway in human cells and illustrate the importance of structural elements, and their context, in potentiating OAS1 activity.

Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem-loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2α subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.

Use of a Grant Writing Class in Training PhD Students.

A well-written application for funding in support of basic biological or biomedical research or individual training fellowship requires that the author perform several functions well. They must (i) identify an important topic, (ii) provide a brief but persuasive introduction to highlight its significance, (iii) identify one or two key questions that if answered would impact the field, (iv) present a series of logical experiments and convince the reader that the approaches are feasible, doable within a certain period of time and have the potential to answer the questions posed, and (v) include citations that demonstrate both scholarship and an appropriate command of the relevant literature and techniques involved in the proposed research study. In addition, preparation of any compelling application requires formal scientific writing and editing skills that are invaluable in any career. These are also all key components in a doctoral dissertation and encompass many of the skills that we expect graduate students to master. Almost 20 years ago, we began a grant writing course as a mechanism to train students in these specific skills. Here, we describe the use of this course in training of our graduate students as well as our experiences and lessons learned.

A Novel Motif for S-Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis.

Capreomycin is a potent ribosome-targeting antibiotic that is an essential component of current antituberculosis treatments, particularly in the case of multidrug-resistant Mycobacterium tuberculosis (Mtb). Optimal capreomycin binding and Mtb ribosome inhibition requires ribosomal RNA methylation in both ribosome subunits by TlyA (Rv1694), an enzyme with dual 2'-O-methytransferase and putative hemolytic activities. Despite the important role of TlyA in capreomycin sensitivity and identification of inactivating mutations in the corresponding Mtb gene tlyA, which cause resistance to capreomycin, our current structural and mechanistic understanding of TlyA action remains limited. Here, we present structural and functional analyses of Mtb TlyA interaction with its obligatory co-substrate for methyltransferase activity, S-adenosyl-l-methionine (SAM). Despite adopting a complete class I methyltransferase fold containing conserved SAM-binding and catalytic motifs, the isolated TlyA carboxyl-terminal domain exhibits no detectable affinity for SAM. Further analyses identify a tetrapeptide motif (RXWV) in the TlyA interdomain linker as indispensable for co-substrate binding. Our results also suggest that structural plasticity of the RXWV motif could contribute to TlyA domain interactions, as well as specific recognition of its two structurally distinct ribosomal RNA targets. Our findings thus reveal a novel motif requirement for SAM binding by TlyA and set the stage for future mechanistic studies of TlyA substrate recognition and modification that underpin Mtb sensitivity to capreomycin.

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization.

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m(1)A1408) methyltransferase ('CacKam'). The apo CacKam structure closely resembles other m(1)A1408 methyltransferases within its conserved SAM-binding fold but the region linking core ß strands 6 and 7 (the 'ß6/7 linker') has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m(1)A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the ß6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.

Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase.

The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m1A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA ß2/3 linker collectively are critical for docking of NpmA on a conserved 16S rRNA tertiary surface. In contrast, other NpmA regions (ß5/ß6 and ß6/ß7 linkers) contain several residues critical for optimal positioning of A1408 but are largely dispensable for 30S binding. Our data support a model for NpmA action in which 30S binding and adoption of a catalytically competent state are distinct: docking on 16S rRNA via the ß2/3 linker necessarily precedes functionally critical 30S substrate-driven conformational changes elsewhere in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Our results provide a molecular framework for aminoglycoside resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.

A novel RNA molecular signature for activation of 2'-5' oligoadenylate synthetase-1.

Human 2'-5' oligoadenylate synthetase-1 (OAS1) is central in innate immune system detection of cytoplasmic double-stranded RNA (dsRNA) and promotion of host antiviral responses. However, the molecular signatures that promote OAS1 activation are currently poorly defined. We show that the 3'-end polyuridine sequence of viral and cellular RNA polymerase III non-coding transcripts is critical for their optimal activation of OAS1. Potentiation of OAS1 activity was also observed with a model dsRNA duplex containing an OAS1 activation consensus sequence. We determined that the effect is attributable to a single appended 3'-end residue, is dependent upon its single-stranded nature with strong preference for pyrimidine residues and is mediated by a highly conserved OAS1 residue adjacent to the dsRNA binding surface. These findings represent discovery of a novel signature for OAS1 activation, the 3'-single-stranded pyrimidine (3'-ssPy) motif, with potential functional implications for OAS1 activity in its antiviral and other anti-proliferative roles.