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Deciphering RNAi machineries required for miRNAs Cell-to-Cell Transfer in Mammals

Final Report Summary - DRIMTIM (Deciphering RNAi machineries required for miRNAs Cell-to-Cell Transfer in Mammals)

Small RNAs are key post-transcriptional regulators of eukaryotic gene expression. Among the most fascinating aspects of small RNAs is their ability to cross cell boundaries owing to their non-cell-autonomy. Recently, our laboratory demonstrated that 21-24bp siRNAs could act as mobile silencing signals in Arabidopsis. Interestingly, in C. Elegans, systemic silencing requires SID1, a transmembrane channel through which endogenous double-stranded RNA may exert their effects in adjacent cells. In mammals the characterization of functional SID1 homologue, and of miRNAs embedded in secreted exosomes, are good evidences of the conservation of this small RNA systemic mechanism. The possibility that miRNAs might act non-cell autonomously in mammals raises the question of how this process might be regulated? A first possibility for regulated miRNA movement entails that it might mostly occur between compatible “emitting” and “receiving” cells. This might be achieved via qualitatively differences in miRNAs effector complexes or, in localization as well as in the shear availability of transporter systems. A second, non-mutually exclusive possibility is that release of miRNA through membrane might be highly polarized. The recent identification, in our laboratory, of a requirement of the multi-vesicular bodies machineries for the assembly of miRNA effector complexes would support this idea. Actually, both mechanisms could be used in specialized cell types such as secreting epithelia to direct the selective release of miRNAs either along the epithelial cell layer, or in body fluids. The DRIMTIM project aimed at providing functional evidences for cell-to-cell movement of small RNA in mammals using cancer cells and the mammary gland system as models.

First, an important aspect of DRIMTIM was the development of innovative cellular tools allowing to track functional transfer of miRNAs. The demonstration of functional miRNA transfer is a challenging task that requires (1) the capacity to monitor miRNA level at the cellular level using sensitive and specific miRNA sensors, and (2) a cellular system allowing to eliminate the contribution of endogenous miRNA in the recipient cells. We initially showed that only a small subset of breast cancer cell lines are competent for either emitting or receiving mobile miRNA. Building on a new and innovative positive miRNA sensor strategy, we achieved the generation of a miR-21 fluorescent posisensor cell line in which miR-21 miRNA in inactivated at the genomic level. When used as a recipient cells, this sensor line allows for sensitive and quantitative monitoring of functional miR-21 transfer events using flow cytometry or fluorescent-microscopy. Using this unique cellular tool, we are currently screening for the capacity of a diverse set of cell lines, or biofluids, to mediate efficient extracellular miR-21 transfer.

Second, DRIMTIM notably focused on identifying molecular heterogeneity of RNA silencing complexes among different mammalians cell as it is currently hypothesized that key licensing factors might be responsible for the capacity of cells to either emit or receive small RNAs. Indeed, we identified several factors whose expression is variable among cell lines either quantitatively (e.g. SID-1 factors) or qualitatively (e.g. Argonaute). We notably discovered isoforms of Argonaute proteins that could directly participate in the capacity of cells to exchange small RNAs. We are currently testing if the expression of SID-1 factors or Argonaute isoforms correlates with the capacity of cells to emit miR-21 using our posisensor assay.

Finally, DRIMTIM aimed at identifying and characterizing in vivo mechanisms for which miRNA transfer is likely to be functionally relevant. Several mammalian biological fluids are know to contain extracellular miRNAs but their physiological relevance is far from being demonstrated. Here we show that mammalian milk contains very abundant microRNAs and RNA silencing factors that could function either as paracrine signaling molecules in the mammary gland, or as a long-distance communication system between mother and child. We notably tested this last hypothesis using miRNA knockout mouse models, and were able to show that indeed, milk miRNA are transferred in mouse pups during breastfeeding, thereby establishing the first proof of evidence of miRNA between mammalian organisms. We are currently conducting experiments aiming at demonstrating the function and stability of these milk miRNAs in pups intestine.