Investigating cell biological aspects of microRNAs-mediated silencing
Zusammenfassung der Projektergebnisse
During the time this project was financed by the DFG, advances were made toward its completion according to the three specific aims that were originally defined. According to the first aim of the project, cell lines were generated that stably express inducible reporters useful for the study of miRNA expression. As compared to the original approach that was proposed, the new approach allows an easier selection of clonal cell lines as well as a ore rigorous comparison between different cell lines as the expression cassette is always introduced as a single copy in the same genomic locus. The cell lines were characterized and behave as they should theoretically perform. Thus the first aim of the project can be considered as completed as the making of further cell lines is now a routine procedure. The second aim of the project was to gain more insight of the localization of the miRNA-associated machinery. The need to study the endogenous proteins and not tagged versions thereof was shown. Several pools of endogenous Ago2 could be observed in biochemical fractionation experiments, one of which physically associated to ER membranes. First experiments could show that the cell lines generated can be used to monitor the localization, in living cells, of the reporter mRNA they express by using the MS2-tag system. Moreover, preliminary experiments showed that endogenous Ago2 can be analyzed by electron microscopy using available antibodies. More work will be needed to bring aim 2 to completion. This will involve further use of the MS2 system to define the localization of the mRNA reporters at different time points after their induction, optimizing the EM procedure, and analyzing the distribution of the mRNA reporters in the fractionation experiments. The main goal will be to try and find the functional significance of the different pools of Ago2. Within this line, efforts will be made to set up and use the correlative immunofluorescence/electron microscopy strategy planned in the original proposal. The third aim was to analyze a potential connection between the cytoskeleton and endomembranes dynamics, and the miRNA pathway. The gathered so far speak again such a connection, or at least if such a connection exist it is not critical for miRNA function. Indeed, treating cells with drugs that completely destroy microtubules or the actin network had no effect on the repression of reporter mRNA. Similarly, drugs disrupting defined steps of the secretory pathway also showed no effect. Moreover, two genes identified in genome-wide screens as miRNA-pathway regulator, and normally involved in microtubules dynamics and Golgi structure integrity, showed no effect in our reporter assay. To complete aim 3, a contribution of the cytoskeleton or endomembranes to the localization of mRNA targets of miRNAs, a process that was shown to be a consequence of but not necessary to miRNA-mediated repression will be investigated. Altogether, the project has progressed according to plan, and is advancing to completion.
