Co-translational mRNA degradation in Drosophila cells

Over the past decade miRNAs emerged as key regulators of gene expression. It is well established that animal miRNAs bind their target mRNA and inhibit its translation and/or accelerate mRNA turnover. In the proposed project we plan to investigate the intertwined relation of mRNA translation and degradation and the consequences for the miRNA response in Drosophila cells. Recent studies have demonstrated the possibility of co-translational mRNA degradation in yeast. Our own preliminary results in Drosophila cells demonstrate the co-migration of factors of the mRNA decay machinery and miRNA effector components with polysomes. Thus, we will first investigate whether normal/bulk mRNAs can be degraded co-translational in Drosophila cells. Secondly, we will investigate the co-translational degradation of mRNAs targeted by miRNAs. In particular we will investigate potential differences between translation dependent and independent mRNA degradation by miRNAs. Thirdly, we propose to investigate how widespread translation dependent mRNA degradation is on a genome-wide level in Drosophila cells. The results of this project will significantly contribute to our understanding of the general mRNA decay pathway and the miRNA-mediated mRNA decay in particular. While the initial studies will be done in Drosophila cells, the results will be important for studying these pathways in higher eukaryotes. Our long-term goal is to identify which mRNAs are co-translational degraded and identify the determinants for co-translational mRNA degradation.

The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors including small non-coding RNAs. While it is well established that translation and mRNA degradation are tightly coupled, it is still not clear where in the cell mRNA degradation takes place. In this study, we investigated the possibility of mRNA degradation on the ribosome in Drosophila cells. Using affinity purification methods ribosome complexes can be isolated out of Drosophila cell lysates. We used the method to isolate mRNAs from purified ribosomal complexes and prepared mRNA libraries of decapped mRNA degradation fragments. Subsequently, the RNA libraries were subjected to HiSeq analysis. Interestingly, 93 % of decapped mRNA fragments (approx. 12,000 different kinds of mRNA) could be detected with the same relative abundance on ribosome complexes as in cell lysates. Thus the majority of mRNA degradation fragments found in these cells can be detected on ribosome complexes. This included mRNAs, which were upregulated in AGO1 knockdown cells and therefore might be regulated by miRNAs. The results of the HiSeq analysis were verified using qRT-PCR analysis of some of the transcripts. In summary the findings of this research project strongly indicate the association of the majority of bulk mRNAs and mRNAs targeted by miRNAs with the ribosome during their degradation. This further suggests that the ribosome is a major site for mRNA degradation.