The scientific goal of the laboratory is to study the events that determine the molecular ‘fate’ of mRNA in the cytoplasm. We focus on the mechanisms that regulate the removal of structures that protect mRNA from degradation: the poly(A) tail at the 3′-end, in a process known as deadenylation, and the 7-methylguanylate cap at the 5′-end, or decapping. We wish to understand what determines the specificity of these processes and their integration with other stages of the gene expression pathway.

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Our principal approach to studying these molecular events is to reconstitute the relevant regulatory macromolecular complexes from highly purified recombinant components and to study them in vitro. To this end, we employ biochemical assays and biophysical approaches to study the interactions between the components using the instrumentation and facilities available at the Biophysical Resource, part of the Center for Structural Biology. To gain a deeper appreciation of the molecular mechanism, we extensively collaborate with our colleagues at the Center for Structural Biology to determine high-resolution cryoEM structures by single-particle analysis using the data collected on the Titan Krios and Talos Arctica microscopes. Among our recent achievements is the reconstitution of the complete human CCR4-NOT deadenylation complex and the entire network of principal human decapping factors. These unique biochemical tools and reagents enable us to study the 5′-to-3′ mRNA decay pathway with unprecedented precision and control. 

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To relate biochemical and structural insights to function, we test our derived hypotheses directly in human cells; for example, by complementing different variants in cell lines where the expression of individual molecular factors has been knocked out by CRISPR gene editing. This approach has proved very powerful in validating and mapping interactions in vivo. We use high-throughput short-read sequencing with bioinformatic analysis to survey cellular transcriptome and translatome dynamics. We also employ long-read direct RNA sequencing approaches to study mRNA metabolism using Oxford Nanopore instruments available at the RNA Biology Laboratory. A long-standing goal of the laboratory is to achieve sufficient understanding to rebuild entire functionally-relevant regulatory processes of mRNA decay in a test tube. Such a platform offers a unique toolkit to optimize mRNA-based therapeutics for stability and expression and to introduce new design features to target cancer more effectively.

Relevance to Cancer

Dysregulation of posttranscriptional control contributes to autoimmune disease, neurodegeneration, and especially toward oncogenic transformation. Our research is focused on the fundamental biological discovery, which is vital for progress in addressing urgent challenges of age-related diseases. Improved mechanistic understanding of the turnover and translation of mRNA holds enormous potential to provide immediate new vantage points for therapeutic intervention in tumor progression and metastasis.