Final Report Summary - CIRCADIAN RHYTHMS (Biochemical and behavioral characterization of miRNA-mediated regulation of circadian rhythms in Drosophila.)
The objectives of the proposal were: 1) Characterize the post-transcriptional control of Clk mRNA and determine its importance for circadian timekeeping; 2) Characterize temporally and spatially the expression and function of circadian-relevant miRNAs; 3) Explore new roles for miRNA-mediated regulation in the input, central and output pathways of the circadian pacemaker.
- A description of the work performed since the beginning of the project (and results obtained so far),
The first aim is the core of the present proposal and has been completed. Our work demonstrated a role for the miRNA bantam in regulating circadian rhythms in Drosophila. We showed that this regulation is key for the central circadian regulator Clk. We published these results in Genes and Development (Kadener et al., 2009). A follow up of this work demonstrated that post-transcriptional control of Clk is essential for restricting CLK activity in time and space (Lerner et al., in preparation). Indeed, flies in which this regulation is abolished display wide ectopic expression of CLK and CLK-target genes. Importantly Clk post-transcriptional control also is essential for avoiding transcriptional noise, which can result in big developmental and behavioral aberrations. Flies in which Clk lost their post-transcriptional control have random behavior and random number of the most important circadian neurons (pdf-expressing neurons). We are currently preparing one manuscript describing these exciting results. Finally, we have performed a dsRNA screening for identifying regulators of Clk 3’ UTR and found that a very well characterized kinase strongly regulates clk post-transcriptional control.
In addition, we also addressed our second aim. In our Genes and Development paper (Kadener et al., 2009), we found a group of circadian relevant RNAs. Among them we found bantam, miR14 and miR956 all of which are regulated or regulate the circadian clock. In particular we have found recently that miR956 display circadian oscillations. We generated tools that allow us to overexpress or diminish the levels of miR956 in a spatial restricted way (by the use of UAS-miR956 or miR956 sponges both of which we generated). We have found some candidate mRNAs regulated by miR-956. In addition, we have profiled miRNAs from fly brains at different circadian timepoints. This allows us to find many circadian and light driven miRNAs. A paper presenting these data is currently in press in Frontiers in Molecular Neuroscience (Mazan et al., 2013).
For performing the third aim, we have generated a new tool that allows us to manipulate the activity miRNA pathway in an inducible way. This is a fly expressing a Dcr1 dominant negative protein under the GAL4/UAS system. Using this tool we demonstrated the importance of miRNA function for circadian behavior, especially after many days in constant darkness. We are currently determining the transcriptional profile in these flies in order to determine circadian pathways (other than locomotor activity) affected by this manipulation.
- The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
Our results have been or will be published in international scientific journal and our data (including RNA-seq) will be available to the scientific community. In the past few years we have engaged in several scientific collaboration with European and non-european scientist, including: Nikolaus Rajewsky (MDC, Berlin, Germany), Aviv Regev (Broad Institute, Cambridge, USA), Silvio Rizzoli (Gottengen, Germany), Manuel Garber (University of Massachussets, Worcester, MA), Michael Rosbash (Brandeis University, Waltham, USA), Damian Refojo (Max Plank Psychiatry Institute, Munich, Germany), Achim Kramer (Charite University, Berlin, USA), Ville Hietakangas (University of Helsinki, Finland), Nir Friedman (Hebrew University, Jerusalem, Israel) and Gil Ast (Tel Aviv University, Israel). In addition I have been invited to the JEDI (Young European Drosophila Investigator) and I have participated in their meetings and initiatives in the last three years.
- A description of the work performed since the beginning of the project (and results obtained so far),
The first aim is the core of the present proposal and has been completed. Our work demonstrated a role for the miRNA bantam in regulating circadian rhythms in Drosophila. We showed that this regulation is key for the central circadian regulator Clk. We published these results in Genes and Development (Kadener et al., 2009). A follow up of this work demonstrated that post-transcriptional control of Clk is essential for restricting CLK activity in time and space (Lerner et al., in preparation). Indeed, flies in which this regulation is abolished display wide ectopic expression of CLK and CLK-target genes. Importantly Clk post-transcriptional control also is essential for avoiding transcriptional noise, which can result in big developmental and behavioral aberrations. Flies in which Clk lost their post-transcriptional control have random behavior and random number of the most important circadian neurons (pdf-expressing neurons). We are currently preparing one manuscript describing these exciting results. Finally, we have performed a dsRNA screening for identifying regulators of Clk 3’ UTR and found that a very well characterized kinase strongly regulates clk post-transcriptional control.
In addition, we also addressed our second aim. In our Genes and Development paper (Kadener et al., 2009), we found a group of circadian relevant RNAs. Among them we found bantam, miR14 and miR956 all of which are regulated or regulate the circadian clock. In particular we have found recently that miR956 display circadian oscillations. We generated tools that allow us to overexpress or diminish the levels of miR956 in a spatial restricted way (by the use of UAS-miR956 or miR956 sponges both of which we generated). We have found some candidate mRNAs regulated by miR-956. In addition, we have profiled miRNAs from fly brains at different circadian timepoints. This allows us to find many circadian and light driven miRNAs. A paper presenting these data is currently in press in Frontiers in Molecular Neuroscience (Mazan et al., 2013).
For performing the third aim, we have generated a new tool that allows us to manipulate the activity miRNA pathway in an inducible way. This is a fly expressing a Dcr1 dominant negative protein under the GAL4/UAS system. Using this tool we demonstrated the importance of miRNA function for circadian behavior, especially after many days in constant darkness. We are currently determining the transcriptional profile in these flies in order to determine circadian pathways (other than locomotor activity) affected by this manipulation.
- The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).
Our results have been or will be published in international scientific journal and our data (including RNA-seq) will be available to the scientific community. In the past few years we have engaged in several scientific collaboration with European and non-european scientist, including: Nikolaus Rajewsky (MDC, Berlin, Germany), Aviv Regev (Broad Institute, Cambridge, USA), Silvio Rizzoli (Gottengen, Germany), Manuel Garber (University of Massachussets, Worcester, MA), Michael Rosbash (Brandeis University, Waltham, USA), Damian Refojo (Max Plank Psychiatry Institute, Munich, Germany), Achim Kramer (Charite University, Berlin, USA), Ville Hietakangas (University of Helsinki, Finland), Nir Friedman (Hebrew University, Jerusalem, Israel) and Gil Ast (Tel Aviv University, Israel). In addition I have been invited to the JEDI (Young European Drosophila Investigator) and I have participated in their meetings and initiatives in the last three years.