Proteomic profiling and functional characterization of post-translational modifications of the fission yeast RNA exosome

Abstract

The RNA exosome is a conserved multi-subunit complex essential for processing and degradation of several types of RNAs. Although many of the functions of the RNA exosome are well established, whether the activity of this complex is regulated remains unclear. Here we performed a proteomic analysis of the RNA exosome complex purified from Schizosaccharomyces pombe and identified 39 post-translational modifications (PTMs), including phosphorylation, methylation, and acetylation sites. Interestingly, most of the modifications were identified in Dis3, a catalytic subunit of the RNA exosome, as well as in the exosome-associated RNA helicase, Mtr4. Functional analysis of selected PTM sites using modification-deficient and -mimetic versions of exosome subunits revealed substitutions that affected cell growth and exosome functions. Notably, our results suggest that site-specific phosphorylation in the catalytic center of Dis3 and in the helical bundle domain of Mtr4 control their activity. Our findings support a view in which post-translational modifications fine-tune exosome activity and add a layer of regulation to RNA degradation.

Proximity-dependent biotinylation by TurboID to identify protein-protein interaction networks in yeast

“…Specifically, only Rrp43 was recovered in the TurboID assay of Rrp6, while TurboID assays of Dis3 did not retrieve any of the ten core exosome subunits. This contrasts to our previous AP-MS analysis of Dis3, as these experiments identified all ten subunits of the core exosome (Telekawa et al, 2018). It is unclear at this point why only a single core subunit of the stable and stoichiometric exosome complex was biotinylated by Rrp6 and Dis3 TurboID fusion proteins.…”

“…Accordingly, many proteins that were previously shown to be associated with the RNA exosome by AP-MS were found to be biotinylated in our TurboID assays of Dis3 and Rrp6 (Fig. 4B,C), including the components of the TRAMP complex Mtr4 and Cid14, the nuclear exosome-associated protein Mpp6, and components of the MTREC/NURS complex, namely Mmi1, Cti1, Iss10, and Mtl1 (Egan et al, 2014;Lee et al, 2013;Telekawa et al, 2018;Zhou et al, 2015).…”

“…To demonstrate the feasibility of the TurboID system in yeast, we initially tagged the C-terminal of the protein arginine methyltransferase Rmt3, and of the 3′-5′ exoribonuclease Dis3, with the 35-kDa TurboID biotin ligase in S. pombe. Rmt3 and Dis3 were selected because affinity purification-coupled mass spectrometry (AP-MS) approaches previously revealed a set of known Rmt3-and Dis3-associated proteins (Bachand and Silver, 2004;Telekawa et al, 2018). Western blot analysis using anti-Myc antibody confirmed the successful tagging of both Rmt3 and Dis3 ( Fig.…”

“…Accordingly, no overlap was found between Rmt3 and Rrp6, which localize to different subcellular compartment, namely the cytosol and the nucleus, respectively. Furthermore, the proteins (Telekawa et al, 2018). that overlapped between TurboID assays of Rmt3 and Dis3 are amongst the most abundant proteins in the fission yeast proteome (Fig.…”

“…PDB assays and AP-MS are known to be complementary proteomics approaches that can identify different sets of interactions for the same bait protein (Lambert et al, 2015). Recently, we used AP-MS to identify and characterize post-translational modifications in the S. pombe RNA exosome complex (Telekawa et al, 2018). In total, 282 proteins were identified in the AP-MS analysis of TAPtagged Dis3, whereas TurboID assays with Dis3 identified 44 proteins.…”