The piRNA pathway and its role in the regulation of maternal mRNAs in the Drosophila early embryo.

• Applicant: Dr. Bridlin Barckmann
• Subject Area: Developmental Biology
• Term: from 2013 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 243843236

In Drosophila maternal mRNAs of determinants for embryonic patterning are regulated by their poly(A)tail length. Poly(A)tail elongation leads to translational activation, and poly(A)tail shortening leads to mRNA decay or translational repression. A gradient of Nanos (Nos) protein, resulting from translational regulation of nos mRNA, organizes abdominal segmentation. In the bulk, but not at the posterior pole of the embryo nos mRNA is repressed. This repression is in part achieved, by recruitment of the CCR4-NOT deadenylation complex by the RNA binding protein Smaug to nos mRNA (Zaessinger et al 2006). The host group showed that CCR4-dependent nos mRNA deadenylation also depends on piRNA pathway components and specific piRNAs targeting nos mRNA. They propose that Aub, loaded with piRNAs, target nos mRNA through piRNA/nos mRNA recognition and help recruiting the CCR4-Not complex to deadenylate nos mRNA (Rouget et al 2010). The piRNAs targeting nos mRNA are produced from transposable elements (TE).This implication of the piRNA pathway in regulation of a cellular mRNA is extremely interesting for two reasons: the identification of a new role of the piRNA pathway in gene regulation and, the role of transposable elements in gene regulation trough piRNA (Rouget et al 2010). During the first two years of the project I analyzed the generality of this regulation and identified direct target mRNAs by:1) The analysis of the impact the piRNA pathway has on maternal mRNA regulation. After piRNA mutant transcriptome analysis I show that there is a broad impact of the piRNA pathway on the decay of maternal mRNAs during the maternal-to-zygotic transition.2) The global analysis of mRNAs directly bound by Aub complex with the iCLIP technique (König et al 2010). Importantly, we find Aub binds, in addition to the expected RNA fragments (like piRNAs and TE mRNA), the mRNAs from 583 genes. We have very promising results indicating a general involvement of the piRNA pathway in maternal mRNA degradation in Drosophila. In the last year of the project I want to thoroughly validate these results by:1) Testing the direct involvement of piRNAs in the regulation of mRNAs by the piRNA pathway.2) Studying if, as it is the case for nos mRNA, deadenylation is the mechanism underlying the piRNA pathway-mediated mRNA regulation.3) Identification of Aub-RNA complexes specific for the bulk of the embryo. Aub is present in the bulk of the embryo where it is involved in the degradation of nos mRNA but also in high levels at the posterior pole where nos mRNA is stabilized. This suggests different roles for Aub in these two compartments. I want to perform Aub-iCLIP in osk mutants that do not form pole cells and therefore serves here as an `all-bulk´ embryo model.The results will increase our understanding of the very basic process of organism development, posttranscriptional gene regulation in general and the co-evolution of transposable elements and their host genomes.

• DFG Programme: Research Fellowships
• International Connection: France
• Host: Martine Simonelig, Ph.D.