General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells

Abstract

The translation and degradation of mRNAs are two key steps in gene expression that are highly regulated and targeted by many factors, including microRNAs (miRNAs). While it is well established that translation and mRNA degradation are tightly coupled, it is still not entirely 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 polysome profiles and ribosome affinity purification, we could demonstrate the copurification of various deadenylation and decapping factors with ribosome complexes. Also, AGO1 and GW182, two key factors in the miRNA-mediated mRNA degradation pathway, were associated with ribosome complexes. Their copurification was dependent on intact mRNAs, suggesting the association of these factors with the mRNA rather than the ribosome itself. Furthermore, we isolated decapped mRNA degradation intermediates from ribosome complexes and performed high-throughput sequencing analysis. Interestingly, 93% of the decapped mRNA fragments (approximately 12,000) could be detected at the same relative abundance on ribosome complexes and in cell lysates. In summary, our findings strongly indicate the association of the majority of bulk mRNAs as well as mRNAs targeted by miRNAs with the ribosome during their degradation.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01346-14.

ACKNOWLEDGMENTS

We thank E. Izaurralde, S. Newbury, and E. Wahle for antibodies and members of the labs of A. Barta and J. Brennecke for help during library preparation.

This work was funded by the Austrian Science Fund (FWF; grants P22124-B09 and P23884-B09 to S.D. and F43 to M.T.W.) and the Vienna Science and Technology Fund (WWTF; LS09-044 to S.D.).