A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping

Nucleic Acids Res. 2017 Jun 20;45(11):6911-6922. doi: 10.1093/nar/gkx353.

Abstract

Cellular liquid-liquid phase separation (LLPS) results in the formation of dynamic granules that play an important role in many biological processes. On a molecular level, the clustering of proteins into a confined space results from an indefinite network of intermolecular interactions. Here, we introduce and exploit a novel high-throughput bottom-up approach to study how the interactions between RNA, the Dcp1:Dcp2 mRNA decapping complex and the scaffolding proteins Edc3 and Pdc1 result in the formation of processing bodies. We find that the LLPS boundaries are close to physiological concentrations upon inclusion of multiple proteins and RNA. Within in vitro processing bodies the RNA is protected against endonucleolytic cleavage and the mRNA decapping activity is reduced, which argues for a role of processing bodies in temporary mRNA storage. Interestingly, the intrinsically disordered region (IDR) in the Edc3 protein emerges as a central hub for interactions with both RNA and mRNA decapping factors. In addition, the Edc3 IDR plays a role in the formation of irreversible protein aggregates that are potentially detrimental for cellular homeostasis. In summary, our data reveal insights into the mechanisms that lead to cellular LLPS and into the way this influences enzymatic activity.

MeSH terms

  • Endoribonucleases / chemistry
  • Endoribonucleases / metabolism
  • Liquid-Liquid Extraction
  • Protein Interaction Maps
  • RNA Processing, Post-Transcriptional
  • RNA Stability
  • RNA, Fungal / chemistry
  • RNA, Fungal / isolation & purification*
  • RNA, Fungal / metabolism
  • RNA, Messenger / chemistry
  • RNA, Messenger / isolation & purification*
  • RNA, Messenger / metabolism
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / metabolism

Substances

  • Dcp2 protein, S pombe
  • Edc3 protein, S pombe
  • RNA, Fungal
  • RNA, Messenger
  • Schizosaccharomyces pombe Proteins
  • mRNA decapping enzymes
  • Endoribonucleases