microRNA Biology

The last decade in biomedical sciences has highlighted unanticipated layers of genome regulation by non-coding RNAs. In particular, microRNAs (miRNAs) have emerged as critical regulators of gene expression in development and disease. miRNAs are ~22 nucleotide endogenous non-coding RNAs that bind to specific miRNA response elements (MREs) in target RNAs, and post-transcriptionally silence gene expression primarily via RNA decay or translational inhibition. Hundreds of miRNAs have been identified in various species, and it is estimated that they collectively have the potential to target nearly two thirds of all mRNAs. However, despite rapid progress in understanding the molecular mechanisms underlying miRNA biogenesis and mechanisms of action, the biological functions of most miRNAs remain elusive at an organismal level. Our goal is to elucidate functions of non-coding regulatory RNAs in animal development, understand their contribution to certain human pathological conditions, and uncover the molecular mechanisms governing miRNA target recognition and silencing. These studies take advantage of a multi-disciplinary experimental platform combining versatile transgenic technologies, RNA biochemistry, molecular biology, bioinformatics, advanced imaging, high-throughput genomic tools, and cutting-edge genome engineering technologies.

miRNA
Sagittal bisection through the Drosophila adult thorax showing the indirect flight muscles (IFM) stained for F-actin (left panels). High resolution structure of IFM myofibrils stained for F-actin (green) and myosin (red) (right panels). Silencing the activity of a single microRNA causes dramatic muscle phenotypes.

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Synthetic Biology

Cellular reprogramming using synthetic gene networks offers much promise as a novel therapeutic tool. Underpinning this idea is the need to generate versatile synthetic devices capable of precise assessment of cellular state and regulation of therapeutic actuation. A central theme in our group is to repurpose the functionality contained within RNA molecules to develop molecular devices capable of rewiring cellular behavior. Attaining this goal requires a rational design process and in vitro evolution strategies, to assemble RNA-based logic gates into biological computers activated by endogenous triggers. miRNA expression profiles can serve as unique signatures differentiating cells based on type as well as physiological state (i.e. disease state), and could form an ideal input for such systems. The resulting synthetic devices have potential for widespread applications ranging from basic tools for miRNA research, to components in targeted diagnostic and therapeutic strategies. This work falls under the broad spectrum of synthetic biology, encompassing fields as diverse as biomedical sciences, engineering and computer sciences.

synBio
A prototype ribozyme-based synthetic device capable of discriminating cell types based on their microRNA expression profile.

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Genome Engineering

The recent advent of sophisticated genome engineering technologies including transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (CRISPR/Cas9), revolutionized biomedical research and created an unprecedented exploratory landscape for basic research, disease studies and therapeutic applications. Our group employs these technologies to answer complex biological and disease questions in a variety of model systems, while continuing to develop innovative dimensions of these tools aimed to advance their in vivo versatility. We have recently developed a powerful experimental platform, which takes advantage of genome engineering to enable rapid interrogation of physiologically relevant MREs and assess their activity in intact biological systems. Using this approach we showed that the activity of MREs is influenced by miRNA : target stoichiometry, and loss of MREs can result in dramatic phenotypes during development. We are currently expanding this experimental framework to multiplex high-throughput analysis of MRE activity in cultured cells. These studies promise to engender unprecedented insight into the principles and rules governing miRNA target selection in the context of a regulatory cellular network. In parallel, we continue to develop new dimensions of genome engineering technologies aimed to advance their in vivo versatility.

GE
CRISPR-MRE: a novel approach for multiplex interrogation of miRNA reponse elements activity.

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