Ribosome profiling reveals a functional role for autophagy in protein translational control

Autophagy promotes protein degradation, and therefore has been proposed to maintain amino acid pools to sustain protein synthesis during metabolic stress. To date, how the autophagy pathway influences the protein translational landscape in mammalian cells remains unclear. Here, we utilize ribosome profiling to delineate the effects of genetic ablation of the autophagy regulator, ATG12, on protein translational control. In mammalian cells, genetic loss of autophagy does not impact global rates of cap dependent translation, even under starvation conditions. Instead, autophagy supports the translation of a subset of mRNAs with complex 5’UTR structures, enriched for cell cycle control and DNA damage repair. In particular, we demonstrate that autophagy enables the translation of the DNA damage repair protein BRCA2, which is functionally required to attenuate DNA damage and promote cell survival in response to PARP inhibition.  Overall, our findings illuminate a new role for autophagy directing the protein translational landscape.

Methods
Immortalized mouse embryonic fibroblasts of the genotype Atg12f/f;CagCreER+ were grown in DMEM supplemented with 10% serum. 5 days prior to the experiment, half of the cells were treated with 2uM 4-hydoxytamoxifen for three consecutive days to induce deletion of Atg12. 

Ribosome profiling experiments were performed using the ARTseq Ribosome profiling kit (Epicentre). Briefly, immortalized Atg12f/f or Atg12KO MEFs were maintained in control media (DMEM + 10% fetal bovine serum) or starved in HBSS for 2 hours. Following the starvation period, cycloheximide made fresh to 50mg/ml in Ethanol for each experiment was added at a final concentration of 100ug/ml.  Then, cells were collected in PBS and preparation of the samples was performed according the ARTseq Ribosome profiling kit manufacturer’s instructions. RNA extraction by Trizol LS (Ambion), rRNA depletion via RiboZero Gold (Epicentre), and quality and quantity of small RNA and DNA assayed using Agilent High Sensitivity Small RNA kit and DNA kit respectively (Agilent). 

Sequencing was performed at the UCSF sequencing core on Illumina HiSeq2000 as single reads at 150nt length, and analysis of reads was performed using Babel (Olshen et al, 2013) with alignment to mm10.protein_coding.ensembl_76. Subsequent analysis of the processed data was performed in R.