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My long-term research interest is to elucidate the role of arginine methylation in the regulation of post-transcriptional mechanisms, with a focus on how these novel molecular pathways are deregulated in human pathologies, including spinal muscular atrophy (SMA) and cancer. Below is a brief description of the three major ongoing projects in the laboratory currently.
Project #1: A Role for Arginine Methylation in Spinal Muscular Atrophy.
(Funded by CIHR until 2013 and Families of SMA Canada through 2012)
Context and Rationale There is presently no cure for spinal muscular atrophy (SMA), a genetic neuromuscular disorder that specifically affects lower amotoneurons in the spinal cord. SMA is the leading genetic killer of young children, with a prevalence of 1/6000, and is caused by disruption of a gene named Smn. Despite a large body of work towards clarifying SMN function in all cells, the molecular defect leading to motoneuronspecific pathologies and development of SMA remains unknown. Motoneurons from SMA mice exhibit normal survival in culture, but reduced axon growth, correlating with mislocalization of bactin mRNA in distal axons. In agreement with this observation, SMN is found in granular foci in axons, although the precise nature of these granular bodies as well as the axonal function of SMN remains to be determined.
My work has demonstrated that the Tudor domain of the SMN protein is a "sensor" of arginine methylation in cellular proteins. Importantly, naturallyoccurring mutations in the Tudor domain, that lead to a loss of this "methylsensing" capacity, are found in human patients with severe cases of SMA, underscoring the functional relevance of this domain in the etiology of the disease. We have identified novel SMN Tudor domain interacting proteins from spinal cord tissues, and found that many of these proteins are known components of neuronal RNA granules. These specialized ribonucleoprotein (RNP) complexes are responsible for the transport and localized expression of mRNAs in axons of neuronal cells. Based on these observations, we hypothesize that SMN participates in the assembly and function of neuronal RNA granules.
Specific Objectives and Methodology We will use a unique motoneuronlike cell line that recapitulates SMA axonal defects, by targeting the 3' UTR of the endogenous SMN mRNA with stably expressed short hairpin RNAs (shRNAs). This system will enable us to study naturallyoccurring Tudor mutations found in human patients with severe type I SMA. Our experimental approaches will also be complemented with primary motoneuron cultures and tissues from established SMA mouse models. We are also using a quantitative mass spectrometry approach, termed "SILAC (Stable Isotope Labeling by Amino acids in Cell culture)", to study SMA. This approach is used to characterize the composition and stoichiometry of the SMN complex in the nuclear, cytoplasmic, and axonal compartments of motoneurons, at different time points during differentiation. Since we have previously shown that arginine methylated proteins are often misregulated in SMA cells, we will also use a variation of the SILAC approach, termed "Heavy methyl SILAC", that will permit the direct and quantitative analysis of endogenous proteins levels AND methylation status in normal vs SMA motoneurons.
Project #2: The Contribution of Specific PRMTs and Their Substrates to Breast Cancer (Funded by 'The Cancer Research Society' until 2011)
Context and Rationale Recent studies strongly suggest a role for arginine methylation in oncogenesis: (i) arginine methylation of specific proteins by PRMT6 correlates with cellular transformation and tumor metastatic potential; (ii) PRMT1 was shown to be an essential component of a novel mixed lineage leukemia oncogenic transcriptional complex; (iii) PRMT5 expression is upregulated in mantle cell lymphoma and this correlates with increased anchorageindependent cell growth; (iv) AS1411, a drug currently in clinical trial for treatment of various cancers, was recently shown to act by modifying the intracellular localization and activity of PRMT5; (v) we have found that a strictly cytoplasmic alternatively spliced isoform of PRMT1, PRMT1v2, is overexpressed in a number of breast cancer cell lines, and (vi) we have now observed that CARM1, PRMT5, 6 and 7 are also upregulated in breast cancer cells, correlating with arginine methylation profiles of several proteins being perturbed. We have shown that PRMT1 isoforms have distinct profiles of interacting proteins/substrates, many with potential implications in breast cancer biology.
Hypothesis and Specific Objectives We hypothesize that misregulation of PRMTs and arginine methylation pathways contributes to the etiology of breast cancer. This project will first use highdensity breast tissue arrays in order to rapidly assess the correlation between PRMT expression levels, arginine methylation profiles, and various breast cancer pathological parameters. Secondly, using RNA interference and a number of well established experimental models, we will determine which PRMT(s) are required for breast cancer tumorigenesis, focusing initially on PRMT1v2 (which we have found is specifically overexpressed in breast cancer cell lines and solid tumors). Finally, a novel quantitative proteomics approach (SILAC) will be used to identify in an unbiased fashion the proteins that are aberrantly arginine methylated in breast cancer cells.
Significance Continued efforts to increase knowledge of breast cancer diagnosis and etiology is essential to answer the need for improved prevention and treatment. This work has the potential to uncover novel pathways involved in cellular transformation and breast tumorigenesis.
Project #3: The role of methyl-binding protein TDRD3 in breast cancer (Funded through Canada Research Chair until 2015)
Context and Rationale - Breast cancer is one of the most common cancers affecting Canadian women. One in nine (11%) Canadian women are expected to develop breast cancer during her lifetime, and on average, 100 Canadian women will die of breast cancer every week. We are interested in understanding the cellular role(s) of arginine methylation, with a focus on its implication in human pathologies, including breast cancer. Interestingly, TDRD3, a protein that we have been the first to characterize, has been identified amongst genes whose high expression has a strong predictive value for poor postoperative prognosis of estrogen receptornegative breast cancers. Our initial work on TDRD3 has demonstrated that its Tudor domain, can serve as a protein module recognizing methylated arginines in proteins, although its cellular function remains unknown. We have more recently further characterized TDRD3 and found that it relocalizes to cytoplasmic Stress Granules (SGs) in response to various cellular stresses.
These cytoplasmic foci are sites of mRNA triage that promote cell survival following exposure to stress stimuli. The hypoxic core of solid tumors represents a pathophysiologic setting where SGs may contribute to tumor cells survival and resistance to chemotherapeutic agents.
Hypothesis: TDRD3, mainly through its role in stress granules, plays a role in promoting late hallmarks of breast cancer pathogenesis, including tumor cells survival, resistance to therapy, and metastasis.
post-transcriptional regulatory mechanisms, neuromuscular, diseases, arginine methylation, Spinal Muscular Atrophy, Myotonic Dystrophy, Fragile X Syndrome, ALS