Conn Lab Research
Our lab uses a broad array of biochemical, structural biology and other state-of-the-art approaches to dissect the structures and functions of biomedically important RNA molecules and their protein binding partners. Current topics include:
Non-coding RNA-mediated regulation of host cell innate immune proteins
Our lab has long-standing interests in the structure and activity of viral non-coding RNAs, such as Adenovirus VA RNAI – an essential, pro-viral RNA best known for inhibition of the double-stranded (ds)RNA-activated protein kinase (PKR). More recently, we have begun new studies that aim to determine which features of simple dsRNAs and larger, complex viral and cellular non-coding RNAs are important for regulation of a second innate immune protein, oligoadenylate synthetase 1 (OAS1).
Current projects include:
Non-coding RNA mediated control of PKR. Our early work defined the stabilities and roles of the conserved domains within the adenoviral non-coding transcript VA RNAI , best known for its inhibitory action against PKR. A remarkable finding was that the entire Terminal Stem could be deleted without loss of activity (whereas smaller deletions were detrimental), complementing the parallel discovery that VA RNAI is similarly processed in the cell by Dicer. Our most recent studies have defined the minimal requirements for PKR inhibition by VA RNAI, offering a satisfying explanation for why VA RNAs from different serotypes are equally effective despite their wide variation in sequence and length (Wilson et al. J. Biol. Chem, 2014).
Our current work is focused on understanding the contributions of each PKR domain and the interdomain linker to inhibition by human viral non-coding RNAs, as well as the structure and activity of a recently identified human cellular non-coding RNA regulator of PKR (nc886).
Molecular mechanisms of RNA-mediated regulation of OAS1. In a new direction to our studies of RNA-mediated regulation of innate immune system proteins, we recently identified a novel signature for activation of the enzyme oligoadenylate synthetase 1 (OAS1). In response to dsRNA, OAS1 produces 2’-5’-linked oligoadenylate second messengers for which the only known target is the latent ribonuclease, RNase L. Activation of the OAS/RNase L pathway triggers a program of cellular and viral RNA degradation designed to halt protein synthesis in the infected cell. We found that a single-stranded RNA motif (termed 3’-ssPy) can potentiate OAS1 activation by diverse RNAs, including a short model dsRNA duplex and several structured viral or cellular non-coding RNAs (Vachon et al. Nucleic Acids Res., 2015). Our current studies will extend this initial finding to define the “rules” that govern potent OAS1 activation by both simple dsRNAs and larger, viral and cellular non-coding RNAs. Additionally, in collaboration with Dr. Anice Lowen at Emory, we aim to uncover the potential impacts of motifs like 3’-ssPy on the activation of the OAS/RNase L pathway in the context of cellular infection.
RNA modification and bacterial antibiotic resistance
Rising antibiotic resistance among human pathogenic bacteria is a major contemporary healthcare problem. We are studying several examples of how bacterial antibiotic resistance can arise through chemical modification (methylation) of the ribosomal RNA (rRNA) antibiotic binding site. This mechanism of resistance is predominant among antibiotic-producing bacteria, but has also more recently been more recently identified as an acquired form of resistance in diverse human pathogens. Such modifications of the drug binding site can confer exceptionally high-level resistance and broad specificity to a given class of antibiotics, making them a significant potential new threat.
Current projects include:
Aminoglycoside-resistance 16S rRNA methyltransferases. Aminoglycoside antibiotics typically act by binding and inducing specific conformational changes in the ribosome “decoding center” that result in aberrant protein synthesis. Aminoglycosides have retained potent activity, leading to a reevaluation of their potential utility in the clinic in the face of increasing resistance to many first line drugs. Drug-producing bacteria invariably use 16S rRNA methyltransferase enzymes to modify their ribosomal drug binding site, either at the N7 position of G1405 (m7G1405) or the N1 position of A1408 (m1A1408). Over the last decade or so, the acquisition and spread among human bacterial pathogens of these enzymes has seen them emerge as a serious new threat to the potential future clinical usefulness of aminoglycosides. 16S rRNA modification at G1405/A1408 confers exceptionally high level resistance and, combined, these modifications are capable of blocking the effects of all clinically useful aminoglycosides including the latest generation drugs.
Determining the structures of these enzymes and defining the features which govern their interactions with cosubstrate S-adenosyl-L-methionine (SAM) and 30S substrate, have been a major focus in our lab. We determined the first structures of m1A1408 enzymes with cosubstrate, KamB from the aminoglycoside-producer S. tenebrarius, and NpmA which was earlier identified as the source of treatment failure in a clinical infection (Macmaster et al. Nucleic Acids Res., 2010). More recently, in collaboration with Christine Dunham’s group we presented a major break through with the determination of a first structure of a resistance methyltransferase (NpmA) bound to its 30S substrate (Dunkle et al. PNAS, 2014). This structure revealed the basis for the requirement of mature 30S as substrate, and the molecular details underpinning specific target recognition, including flipping of the target A1408 base into the NpmA active site.
Our on-going studies include efforts to obtain complementary structure-function insights for members of the m7G1405 family, and investigating the molecular mechanisms of action of enzymes from both families. Our long term goal is to exploit the understanding we develop of these enzymes and their target recognition mechanisms to facilitate development of specific inhibitors of these resistance determinants.
The thiostrepton-resistance methyltransferase (Tsr). Tsr is a SpoU/TrmD (SPOUT) family rRNA methyltransferase enzyme which methylates the ribose 2’-OH of adenosine 1067 (A1067) in the bacterial large ribosomal subunit (23S) rRNA to confer high-level resistance to thiostrepton. We previously determined the structure of Tsr in complex with SAM (Dunstan et al. J. Biol. Chem., 2008) and, more recently, identified a critical role for RNA conformational changes in controlling Tsr substrate recognition and activation of methyltransfer activity (Kuiper & Conn, J. Biol. Chem. 2014). Our current goals are to determine the structure of the Tsr-rRNA complex to reveal important new insights into RNA recognition by the SPOUT family of methyltransferases and allow the specific but distinct RNA recognition processes of two proteins (L11 and Tsr) to be directly compared.
Molecular mechanisms of RNA and protein methylations in biology and disease
In collaboration with other labs at Emory and elsewhere, we are investigating the molecular mechanisms and biological impacts of other RNA and protein modification processes.
Current projects include:
tRNA modification by Trm10. In collaboration with Drs. Jane Jackman (Ohio State University) and Lindsay Comstock (Wake Forest University) we are investigating the structural basis for tRNA recognition and modification by yeast and human Trm10 methyltransferase.
Lysine 5 (K5) trimethylation of P. aeruginosa EF-Tu. In P. aeruginosa the translation factor EF-Tu is trimethylated on residue K5 and found on the bacterial surface, where it is proposed to mediate attachment to airway respiratory cells through platelet-activating factor receptor. In collaboration with Dr. Joanna Goldberg (Emory University), whose group discovered this EF-Tu modification and the enzyme responsible (EftM), we are investigating the recognition and modification of EF-Tu by EftM, and the molecular basis of EftM temperature sensitivity which may provide a molecular “off switch” for EF-Tu modification following infection.Read Less