RNA Silencing

Short RNAs, as regulators of cellular function, can impact the maintenance of genomic integrity and stability, on cell growth, differentiation and developmental processes, and on the antiviral RNA-silencing response. RNA silencing refers to small interfering RNA (siRNA)-mediated post-transcriptional gene regulation, resulting in the silencing of viral genes and transgenes. Such interactions involve highly specific, adaptive, mobile, and systemic processes that operate in essence as an RNA-based immune response.

Epigenetic Regulation: Histone Marks

The packaging of DNA within chromosomes, the orderly replication and distribution of chromosomes, the maintenance of genomic integrity, and the regulated expression of genes depend upon nucleosomal histone proteins. Our long-term goals are directed toward gaining structural and mechanistic insights into the functional relevance of histone covalent modification(s). Currently, we are structurally investigating the binding of effectors targeted to specific covalent marks in a context-dependent fashion.

Epigenetic Regulation: DNA Methylation Marks

Methylation of cytosine in the CpG context has pronounced effects on gene expression with DNA methylation patterns established during embryonic development and then faithfully maintained during subsequent somatic cell division. The basic principles underlying the setting up and maintenance of DNA methylation patterns remains an area of intense research, given that perturbation of DNA methylation patterns impacts on a range of human diseases. Current efforts are focused on structure-function studies of writers, readers and erasers of DNA methylation marks and their complexes with unmodified and hemimethylated DNA.

Cytoplasmic Metazoan Nucleic Acid Sensors

Host defense against infection by viral and bacterial pathogens is critically dependent on the initiation and maintenance of the finely tuned primary innate immune response, a rapid protective response that is coupled to subsequent adaptive immunity, thereby providing long-term protection based on immunological memory. The innate immune response is equipped with a number of pattern recognition receptors that detect characteristic microbial components, ranging from unmethylated CpG DNA, double-stranded RNA and 5’-triphosphorylated RNA. Our efforts are primarily focused on the metazoan second messenger cyclic GAMP produced by DNA-activated cyclic GMP-AMP synthase, together with elucidation of the principles underlying activation of STING by cGAMP and targeting of STING by anti-viral small molecules.

Riboswitches and Ribozymes

The role of RNA in information transfer and catalysis highlights its dual functionalities. Our laboratory has a long-standing interest in RNA folding, recognition, and catalysis. We are especially interested in both natural and in vitro selected RNA aptamer-based systems, because they serve as exceptional scaffolds for ligand recognition and catalysis, exhibiting tunable specificities and enantiomeric selectivities. Much of our effort is also focused on mRNA because of its functional importance at the cellular level and because a diverse set of ligand and protein interactions control its transcription, splicing, export, localization, translation, and degradation functions.

Protein-RNA Complexes Mediating Disease Syndromes

A number of cancer-related neurodegenerative diseases are associated with RNA-binding proteins. These include FMRP protein in fragile X mental retardation (FXMR) syndrome and Nova and Hu proteins in paraneoplastic opsoclonus-myoclonus ataxia (POMA) syndrome. These also include the La and Ro60 autoantigens, identified in patients with the autoimmune diseases systemic lupus erythematosus and Sjörgen’s syndrome. More recently, we are investigating protein-RNA complexes involved in alternate splicing, with the initial emphasis on muscleblind MBNL and CUGBP1 proteins that have impact on muscular dystrophy. Additional examples include RNA complexes with STAR family Quaking proteins involved in myelination, with TAP protein involved in nucleocytoplasmic transport, and with the multimeric scaffold of the translin-TRAX endoribonuclease.

Replication of DNA Damage Sites by Bypass Polymerases

Genomic integrity depends critically on the fidelity and efficiency of DNA replication. Processive polymerases can stall at DNA damage sites and translesion synthesis is then dominated by bypass polymerases, involving error-free (mutation-avoiding) or error-prone (mutation-generating) pathways. Our long-term goals are directed toward understanding the molecular interactions that define the mutagenic spectrum of activities associated with replication of damage sites by bypass polymerases. The initial efforts are focused on oxidative damage lesions and their processing by the bypass polymerase Dpo4.

Lipid Transfer Proteins

Lipid transfer proteins are important in membrane vesicle biogenesis and trafficking, signal transduction and immunological presentation processes. The conserved and ubiquitous mammalian glycolipid transfer proteins (GLTPs) serve as potential regulators of cell processes mediated by glycosphingolipids (GSLs), ranging from differentiation and proliferation to invasive adhesion, neurodegeneration, and apoptosis. We have initiated a structural biology program toward defining a framework for understanding how GLTPs acquire and release GSLs during lipid intermembrane transfer and presentation processes. This research has been extended to transfer of ceramide-1-phosphate by its transfer protein CPTP.

Higher Order G-Quadruplex Architectures

It has been widely accepted that DNA can adopt other biologically relevant structures beside the Watson-Crick double helix. Our laboratory has focused its efforts on the structure and recognition of multi-stranded DNA architectures adopted by guanine-rich sequences. Such purine-rich sequences are frequently located within gene regulatory regions and recombination hot spot sites, and as tandem repeats in telomeric, centromeric, and triplet repeat disease sequences. Our emphasis is on discovering the range of topologies adopted by G-quadrulex scaffolds.

Aminoglycoside Antibiotic-RNA Complexes

Aminoglycoside antibiotics are polycationic saccharides that exhibit therapeutic potential against bacterial infections. Functionally they interfere with translation and induce bacterial cell death through site-specific targeting of ribosomal 16S RNA, as well as inhibit viral regulatory protein-RNA interactions. Our research has focused on aminoglycoside antibiotic-RNA aptamer complexes in an effort to deduce common structural principles associated with molecular recognition and encapsidation of the bound aminoglycoside antibiotic by the tertiary fold of the RNA pocket.