Updated project metadata. The RNA exosome is a multi-subunit complex essential for processing and degradation of many classes of RNA. Although many of the functions of the RNA exosome are well established, how the activity of this complex is regulated remains poorly understood. Here we report a comprehensive proteomic analysis of the RNA exosome complex from the fission yeast Schizosaccharomyces pombe and identified 39 post-translational modifications (PTMs), including phosphorylation, methylation, and acetylation. Interestingly, most of the modifications were identified in Dis3 and Rrp6, the catalytic subunits of the RNA exosome, as well as in the exosome-associated RNA helicase, Mtr4. Functional characterization of residues found to be modified in distinct exosome subunits revealed specific substitutions that affected cell growth and RNA exosome functions. Notably, phosphomimetic substitutions of phosphorylation sites Ser-809 and Tyr-814 in S. pombe Dis3 resulted in striking loss-of-function phenotype in vivo. Biochemical analysis of the Ser-809 and Tyr-814 phosphomimetic versions of Dis3 revealed proper assembly in vivo, but a marked decrease in degradation capacity in vitro. Given that Ser-809 and Tyr-814 are positioned in the vicinity of the catalytic centre of Dis3, our data suggest that site-specific phosphorylation of the RNA exosome represents a mechanism to control its activity in vivo.