LOSS OF PRP4K IMPAIRS AUTOPHAGIC FLUX IN DICTYOSTELIUM DISCOIDEUM AND HUMAN CANCER CELLS

Abstract

The pre-mRNA splicing factor 4 kinase (PRP4K) is a haploinsufficient tumour suppressor conserved from Dictyostelium discoideum (D. discoideum) to humans. Here, we describe the first viable knockout (KO) model in D. discoideum, where the complete loss of the prpf4B gene results in developmental phenotypes associated with impaired macroautophagy (hereafter referred to as autophagy), including accumulation of the LC3B homolog Atg8. Using small-hairpin RNA (shRNA) knockdown (KD) PRP4K HeLa and MCF7 cancer cells, we similarly observed increased levels of LC3B, autophagy adapter protein and p62/SQSTM after PRP4K KD cells. Consistent with impaired autophagic flux arising from failed autophagosome-lysosome fusion and reduced autophagosome turnover, we observed reduced quenching of GFP fluorescence in the gold-standard mCherry-GFP-LC3B flux assay in MCF7 cells after PRP4K loss. Collectively, these findings have uncovered a novel role for PRP4K in autophagy that is evolutionarily conserved from amoeba to humans.