In vivoCharacterization of the Critical Interaction between the RNA Exosome and the Essential RNA Helicase Mtr4 inSaccharomyces cerevisiae

Abstract

The RNA exosome is a conserved molecular machine that processes/degrades numerous coding and non-coding RNAs. The 10-subunit complex is composed of three S1/KH cap subunits (human EXOSC2/3/1; yeast Rrp4/40/Csl4), a lower ring of six PH-like subunits (human EXOSC4/7/8/9/5/6; (yeast Rrp41/42/43/45/46/Mtr3), and a singular 3-5 exo/endonuclease DIS3/Rrp44. Recently, several disease-linked missense mutations have been identified in genes encoding the structural cap and core subunits of the RNA exosome. In this study, we characterize a rare multiple myeloma patient missense mutation that was identified in the cap subunit geneEXOSC2. This missense mutation results in a single amino acid substitution, p.Met40Thr, in a highly conserved domain of EXOSC2. Structural studies suggest this Met40 residue makes direct contact with the essential RNA helicase, MTR4, and may help stabilize the critical interaction between the RNA exosome complex and this cofactor. To assess this interactionin vivo, we utilized theSaccharomyces cerevisiaesystem and modeled theEXOSC2patient mutation into the orthologous yeast geneRRP4, generating the variantrrp4 M68T. Therrp4 M68Tcells have accumulation of certain RNA exosome target RNAs and show sensitivity to drugs that impact RNA processing. Additionally, we identified robust negative genetic interactions therrp4 M68Tvariant and RNA exosome cofactor mutants, particularlymtr4mutant variants. This study suggests that theEXOC2mutation identified in a multiple myeloma patient may impact the function of the RNA exosome and provides anin vivoassessment of a critical interface between the RNA exosome and Mtr4.