Wu, Edlyn et al. (2009) International Worm Meeting "MicroRNA-mediated translation repression and deadenylation in C.elegans embryos."

Flamand, Mathieu, Sonenberg, Nahum, Mathonnet, Geraldine, Wu, Edlyn, Fabian, Marc, Duchaine, Thomas, Thivierge, Caroline

[International Worm Meeting, 2009]

MicroRNAs (miRNAs) are small RNAs that play a pivotal role in post-transcriptional gene regulation. Most commonly in animals, these regulatory RNAs target mRNAs by imperfectly binding to complementary sites in 3'' untranslated regions (3''UTR), thereby affecting the translation of the targets, or reducing their stability. Despite the significant roles miRNAs play in various biological processes, the mechanistic details of how they regulate gene expression remain unclear. We present here the first in vitro translation system derived from C.elegans embryos that recapitulates cap- and polyA-dependent translation as well as miRNA-dependent silencing. Using luciferase reporters fused to an artificial 3''UTR containing sites complementary to the maternally contributed mir-35 miRNA family, we observed up to 70% inhibition by mir-35 over a 3-hour translation time-course. Upon addition of 2''-O-methyl oligonucleotide inhibitors for the endogenous mir-35 family, translation of our reporters was restored. To understand how this inhibition occurred, we examined the fate of our reporter mRNAs. Inhibited mRNA reporters became fully deadenylated within the first 60 minutes of incubation with our cell-free extract, while a reporter bearing a mutation in the region complementary to the miRNA seed was not susceptible to inhibition or deadenylation. MiRNA-mediated deadenylation is independent of the translation of the targets as neither ApppN capping of the transcripts, nor the presence of the translation inhibitor cycloheximide inhibited deadenylation. We will describe our efforts to screen for endogenous miR-35 targets via deadenylation and translation inhibition assays. We will further define the genetic requirements for translation repression and processing of target mRNAs by testing extracts that are RNAi-depleted. Our findings indicate that deadenylation plays a prominent role in miRNA-mediated control of gene expression in the C.elegans embryo.

Wu, Edlyn et al. (2009) International Worm Meeting "A cell-free C. elegans embryonic system reveals maternal translation control by the miR-35-42 microRNA family through deadenylation."

Thivierge, Caroline, Flamand, Mathieu, Wu, Edlyn, Sonenberg, Nahum, Wohschlegel, James, Duchaine, Thomas F., Fabian, Marc, Mathonnet, Geraldine

[International Worm Meeting, 2009]

Powerful genetic methods in C. elegans have unraveled numerous translational control phenomena, including microRNA-mediated silencing. Better resolution on the mechanisms of microRNA-mediated silencing requires in vitro translation systems that are currently missing from the C. elegans experimental paradigm. We report here the first C. elegans embryonic extract able to sustain potent 5''-Cap- and 3''-poly(A)- synergic translation initiation of exogenous transcripts. Using a reporter system based on the abundant and maternally loaded miR-35-42 family we recapitulated efficient microRNA-mediated silencing in vitro. We found mRNA reporters to be rapidly and completely deadenylated in a microRNA-dependent manner. Unexpectedly, complete deadenylation did not result in the destabilization of the targeted transcripts up until at least 2 hours post-deadenylation. The stability of the deadenylated intermediate prompted us to propose a model wherein miRNA-mediated silencing of maternally contributed transcripts is reversible via the regulated control of poly(A) tail length. Finally, functional proteomic analysis of the embryonic microRNA-mediated silencing machinery, and the systematic validation in this system is underway and progress will be presented. This work is supported by the Canadian Institute of Health and Research and by the National Sciences and Engineering Research Council of Canada..

Pause, Arnim et al. (2015) International Worm Meeting "Loss of Folliculin confers osmotic stress resistance via AMPK-dependent remodeling of carbohydrate stores in C. elegans."

Coull, Barry, Flamand, Mathieu, Possik, Elite, Vijayaraghavan, Tarika, Ajisebutu, Andrew, Manteghi, Sanaz, Hall, David, Pause, Arnim, van Stensel, Maurice

[International Worm Meeting, 2015]

Mechanisms of adaptation to environmental changes in osmolarity are fundamental for cellular life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. Here we identify a novel AMPK-dependent pathway of resistance to hypertonic stress mediated by the AMPK regulator flcn-1 in C. elegans. FLCN-1 is the worm homologue of the tumor suppressor Folliculin (FLCN), responsible for the Birt-Hogg-Dube hereditary cancer disorder. We show that loss of flcn-1 increases glycogen stores in an AMPK dependent manner and that the glycogen reserves are rapidly degraded upon hypertonic stress, leading to a remarkable accumulation of the organic osmolyte glycerol, promoting resistance to hyperosmotic stress. Importantly, loss of AMPK, glycogen synthase or glycogen phospharylase, which are critical enzymes in glycogen metabolism, strongly suppressed the increased osmotic stress resistance in flcn mutant animals. We further demonstrate that glycerol 3-phosphate dehydrogenase-1 is strongly induced in flcn-1 animals upon hyperosmotic stress and that simultaneous loss of gpdh-1 and gpdh-2 abolished the flcn-1/AMPK-mediated osmotic stress phenotype. Importantly, we show that glycogen accumulates in kidneys from mice lacking FLCN and in kidneys and renal tumors from a BHD patient. Since BHD is a renal hyperproliferation disorder, a mechanism of osmotic stress resistance in kidney hyperosmotic environments might explain tumorigenesis in BHD patients. Overall, our data indicate that FLCN is an evolutionary conserved regulator of glycogen metabolism, that might be acting as a tumor suppressor via AMPK-dependent accumulation of glycogen and organic osmolytes, resulting in an advantageous increase in proliferation and survival in hyperosmotic environments. .