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.
Bacteria and archaea have developed a set of defense mechanisms to protect themselves against invaders such as phages and plasmids. Central among these are CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which directly target the incoming phage/plasmid DNA and or RNA. The defense mechanism of CRISPR-Cas systems can be divided into three stages: (1) spacer acquisition, (2) crRNA (CRISPR RNA) biogenesis and (3) target interference. Based on the architecture of the interference modules, the CRISPR-Cas systems can be broadly grouped into two classes: class 1 systems possess multi-subunit protein complexes termed Cascade, whereas class 2 systems rely on single effector Cas family proteins. Our laboratory is focused on complexes of Cascade and Cas proteins with guide RNAs and target dsDNAs and/or RNAs, as well as evolved anti-CRISPR proteins that silence CRISPR-Cas-mediated cleavage of dsDNA and/or RNA targets.
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.
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. c acid Other areas of interest include cross-talk between DNA and histone methylation, as well as protein-protein and protein-nucleic interactions in RNA-directed DNA methylation.
The biology of histone proteins encompasses their synthesis in the cytosol, nuclear import and incorporation into nucleosomes, as well as subsequent eviction from chromatin, redeposition, storage or degradation. Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. Current efforts are aimed at addressing the specificity underlying histone chaperone binding to histones H3-H4, H2a-H2b and their variants, as well as the topology of the intermolecular interactions.
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.
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.
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.
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.