DOI: 10.7272/q6n877zt
title: Ribosome profiling reveals a functional role for autophagy in protein translational control

description: 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.
description: 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. 
description: Raw data: fastq files are named according to biological
 replicate, total RNA or RNA protected fragment, and condition. RPKK1-4
 indicates which biological replicate the sequencing is from, Total
 indicates all RNA from the sample, RPF means ribosome protected fragment
 from the sample, 1-4 indicates condition where 1 = Atg12f/f in control
 media, 2 = Atg12f/f in starvation (HBSS2h), 3 = Atg12KO in control media,
 4 = Atg12KO in starvation (HBSS2h).  The adapter
 sequence is adapter: AGATCGGAAGAGCACACGTCT Babel: For
 Babel analysis, the raw files are grouped by replicate and condition, and
 compared to each other by condition. Files were read into babel as
 follows: - :sample_name: control_fed_1<br>  
 :rna:<br>     :inputs:<br>     -
 RPKK1Total1_S43_L006_R1_001.fastq.gz<br>     -
 RPKK1Total1_S59_L006_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK1RPF1_S39_L006_R1_001.fastq.gz<br>    
 - RPKK1RPF1_S55_L006_R1_001.fastq.gz - :sample_name:
 control_fed_2<br>   :rna:<br>     :inputs:<br>     -
 RPKK2Total1_S31_L005_R1_001.fastq.gz<br>     -
 RPKK2Total1_S37_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK2RPF1_S27_L005_R1_001.fastq.gz<br>    
 - RPKK2RPF1_S33_L005_R1_001.fastq.gz - :sample_name:
 control_fed_3<br>   :rna:<br>     :inputs:<br>     -
 RPKK3Total1_S23_L004_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK3RPF1_S19_L004_R1_001.fastq.gz
 - :sample_name: control_fed_4<br>   :rna:<br>    
 :inputs:<br>     - RPKK4Total1_S35_L005_R1_001.fastq.gz<br>  
   - RPKK4Total1_S41_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK4RPF1_S47_L006_R1_001.fastq.gz<br>    
 - RPKK4RPF1_S63_L006_R1_001.fastq.gz #2<br> -
 :sample_name: control_starved_1<br>   :rna:<br>    
 :inputs:<br>     - RPKK1Total2_S44_L006_R1_001.fastq.gz<br>  
   - RPKK1Total2_S60_L006_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK1RPF2_S40_L006_R1_001.fastq.gz<br>    
 - RPKK1RPF2_S56_L006_R1_001.fastq.gz - :sample_name:
 control_starved_2<br>   :rna:<br>     :inputs:<br>     -
 RPKK2Total2_S32_L005_R1_001.fastq.gz<br>     -
 RPKK2Total2_S38_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK2RPF2_S28_L005_R1_001.fastq.gz<br>    
 - RPKK2RPF2_S34_L005_R1_001.fastq.gz - :sample_name:
 control_starved_3<br>   :rna:<br>     :inputs:<br>     -
 RPKK3Total2_S24_L004_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK3RPF2_S20_L004_R1_001.fastq.gz
 - :sample_name: control_starved_4<br>   :rna:<br>    
 :inputs:<br>     - RPKK4Total2_S36_L005_R1_001.fastq.gz<br>  
   - RPKK4Total2_S42_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK4RPF2_S48_L006_R1_001.fastq.gz<br>    
 - RPKK4RPF2_S64_L006_R1_001.fastq.gz #3<br> -
 :sample_name: atg_minus_fed_1<br>   :rna:<br>    
 :inputs:<br>     - RPKK1Total3_S45_L006_R1_001.fastq.gz<br>  
   - RPKK1Total3_S61_L006_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - JG01/RPKK1RPF3_S41_L006_R1_001.fastq.gz<br>
     - JG01/RPKK1RPF3_S57_L006_R1_001.fastq.gz -
 :sample_name: atg_minus_fed_2<br>   :rna:<br>    
 :inputs:<br>     - RPKK2Total3_S33_L005_R1_001.fastq.gz<br>  
   - RPKK2Total3_S39_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK2RPF3_S29_L005_R1_001.fastq.gz<br>    
 - RPKK2RPF3_S35_L005_R1_001.fastq.gz - :sample_name:
 atg_minus_fed_3<br>   :rna:<br>     :inputs:<br>     -
 RPKK3Total3_S25_L004_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK3RPF3_S21_L004_R1_001.fastq.gz
 - :sample_name: atg_minus_fed_4<br>   :rna:<br>    
 :inputs:<br>     - RPKK4Total3_S37_L005_R1_001.fastq.gz<br>  
   - RPKK4Total3_S43_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK4RPF3_S49_L006_R1_001.fastq.gz<br>    
 - RPKK4RPF3_S65_L006_R1_001.fastq.gz #4<br> -
 :sample_name: atg_minus_starved_1<br>   :rna:<br>    
 :inputs:<br>     - RPKK1Total4_S46_L006_R1_001.fastq.gz<br>  
   - RPKK1Total4_S62_L006_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK1RPF4_S42_L006_R1_001.fastq.gz<br>    
 - RPKK1RPF4_S58_L006_R1_001.fastq.gz - :sample_name:
 atg_minus_starved_2<br>   :rna:<br>     :inputs:<br>    
 - RPKK2Total4_S34_L005_R1_001.fastq.gz<br>     -
 RPKK2Total4_S40_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK2RPF4_S30_L005_R1_001.fastq.gz<br>    
 - RPKK2RPF4_S36_L005_R1_001.fastq.gz - :sample_name:
 atg_minus_starved_3<br>   :rna:<br>     :inputs:<br>    
 - RPKK3Total4_S26_L004_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK3RPF4_S22_L004_R1_001.fastq.gz
 - :sample_name: atg_minus_starved_4<br>   :rna:<br>  
   :inputs:<br>     - RPKK4Total4_S38_L005_R1_001.fastq.gz<br>
     - RPKK4Total4_S44_L005_R1_001.fastq.gz<br>   :rp:<br>    
 :inputs:<br>     - RPKK4RPF4_S50_L006_R1_001.fastq.gz<br>    
 - RPKK4RPF4_S66_L006_R1_001.fastq.gz
 :babel_tests:<br> - :babel_name: all_groups<br>  
 :groups:<br>   - :group_name: control_fed<br>     :samples: [
 control_fed_1, control_fed_2, control_fed_3, control_fed_4 ]<br>   -
 :group_name: control_starved<br>     :samples: [ control_starved_1,
 control_starved_2, control_starved_3, control_starved_4 ]<br>   -
 :group_name: atg_minus_fed<br>     :samples: [ atg_minus_fed_1,
 atg_minus_fed_2, atg_minus_fed_3, atg_minus_fed_4 ]<br>   -
 :group_name: atg_minus_starved<br>     :samples: [
 atg_minus_starved_1, atg_minus_starved_2, atg_minus_starved_3,
 atg_minus_starved_4 ] autophagy_rp_orf.cov: shows
 number of read counts per mRNA identifier. columns are for each
 sample. <br>  <br> control = Atg12f/f; atg_minus =
 Atg12KO<br> number indicates which biological replicate<br>
 .rna = total RNA ; .rp = RPF autophagy_rp_qcsummary:
 provides quality control information for the sequencing and data analysis.
 Columns present data per sample.  control = Atg12f/f;
 atg_minus = Atg12KO<br> number indicates which biological
 replicate<br> .rna = total RNA ; .rp = RPF
 <br> Reading the Babel outputs in R: see
 autophagy_rp_analysis.R.  All statistical results
 comparing ribosome occupancy between conditions from Babel (between.babel
 analysis) are also presented as .csv files. Column
 names: mRNA_logFC indicates mRNA fold change on log scale between
 conditions; mRNA_pval indicates significance of the mRNA change between
 conditions; pval indicates significance of ribosome occupancy change
 between conditions; FDR indicates false discovery rate of ribosome
 occupancy change between conditions; Direction indicates whether the
 ribosome occupancy was enriched or decreased between conditions. For
 Atg12ff_Fed_v_Atg12KO_Fed.csv and Atg12ff_starved_v_AtgKO_starved.csv: -1
 = ribosome occupancy higher in Atg12KO; 1 = ribosome occupancy higher in
 Atg12f/f. For Atg12ff_Fed_v_Atg12ff_starved.csv: -1 = ribosome occupancy
 higher in starved than fed; 1 = ribosome occupancy higher in fed than
 starved