Multiplex image-based autophagy RNAi screening identifies SMCR8 as ULK1 kinase activity and gene expression regulator

Elife. 2017 Feb 14;6:e23063. doi: 10.7554/eLife.23063.

Abstract

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator.

Keywords: C9ORF72; SMCR8; ULK1 complex; autophagy; biochemistry; cell biology; gene expression; human; protein kinase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy*
  • Autophagy-Related Protein-1 Homolog / metabolism*
  • C9orf72 Protein / metabolism
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line
  • Gene Expression Regulation*
  • Genetic Testing
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Protein Binding
  • RNA Interference

Substances

  • C9orf72 Protein
  • C9orf72 protein, human
  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • SMCR8 protein, human
  • Autophagy-Related Protein-1 Homolog
  • ULK1 protein, human

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.