A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response.

Melanie Blasius,Jiri Bartek,Chunaram Choudhary,Stephen P Jackson,Sebastian A Wagner

doi:10.1101/gad.246272.114

Abstract

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.

Highlights

The cellular DNA damage response (DDR) is controlled by protein post-translational modifications, phosphorylation (Polo and Jackson 2011)
We show that ultraviolet light (UV) exposure leads to MAPKAP kinase-2 (MK2)-mediated RBM7 phosphorylation, triggering 14-3-3 binding and controlling promoter upstream transcripts (PROMPTs) turnover, highlighting PROMPT regulation as a new facet of the DDR
We performed a quantitative proteomic screen to identify UV-induced 14-3-3 interactors (Fig. 1A) by cotransfecting human HCT116 cells with plasmids expressing green fluorescent protein (GFP)-tagged 14-3-3e and 14-3-3z, two of the seven human 14-3-3 isoforms with links to cell cycle control via binding CHK1 and CDC25 (Jiang et al 2003; Dalal et al 2004)

Summary

Introduction

The cellular DNA damage response (DDR) is controlled by protein post-translational modifications, phosphorylation (Polo and Jackson 2011). Reinhardt and Yaffe 2009), which includes MK2-mediated phosphorylation of RNA-binding proteins (Reinhardt et al 2010). Highlighting DDR roles for 14-3-3 proteins, DNA damage-induced CHK1 phosphorylation triggers 143-3 binding and CHK1 nuclear accumulation (Chen et al 1999; Jiang et al 2003).

Results

Conclusion