Project PXD004980

Summary

Title

eIF1 modulates the recognition of sub-optimal translation start sites and steers gene expression control mediated by uORFs.

Description

Alternative translation initiation mechanisms such as leaky scanning and re-initiation potentiate the polycistronic nature of transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stress stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4,000 proteins and 10,000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. The stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with sub-optimal start sites. Affected genes were implicated in energy production and sensing of metabolic stress. Interestingly, knockdown of eIF1 elicited a synergic response from eIF5 and eIF1B.

Sample Processing Protocol

The HAP1 wild type and CRISPR/Cas9 engineered knockout cell lines were obtained from Horizon Genomics GmbH, Vienna. In particular, a single eIF1B knockout clone and two eIF1 knockout clones were acquired (i.e. and eIF1-14bp deletion knock out (eIF1-14bp) and eIF1-265bp insertion knock out (eIF1-265bp)). The human colon cancer cell line HCT116 cells were transfected with either 10nM control si-RNA (si-Ctrl, ON-TARGETplus Non-targeting Control siRNAs: D-001810-01-05, Dharmacon, GE Healthcare Life Sciences) or 10nM si-RNAs targeting eIF1 (si-eIF1, SMARTpool: M-015804-01-0005, Dharmacon, GE Healthcare Life Sciences) using 63ul of HiPerFect (QIAGEN) per 10 cm2 plate. For label-free shotgun proteomics experiments in HAP1 cells, 3 biological replicates of WT cells, eIF1B knockout and both eIF1 knockout clones were prepared. For the label-free shotgun proteomics experiment in HCT116 cells, 2 biological replicates of si-Ctrl cells and si-eIF1 were prepared in parallel with the ribo-seq experiment. 10 million cells per replicate were harvested and lysed by 3 rounds of freeze-thaw lysis in 300µL Gu.HCl lysis buffer (4M Gu.HCl, 50 mM NH4HCO3 pH 7.9). The lysates were sonicated using a Branson probe sonifier and centrifuged for 30 minutes at 3,500g (4 °C). Supernatant aliquot equivalent of 200 µg was transferred to a clean 2 mL-tube and diluted to 1 mg/mL with 4M Gu.HCl + 50 mM NH4HCO3. The protein mixture was further diluted with an equal volume of HPLC grade water followed by a precipitation with 4x volumes of -20 °C acetone for 2 hours at -20°C. Precipitated proteins were collected by centrifugation at 4,000g for 15 minutes (4 °C). Pellets were washed twice with 1 mL of ice-cold 80% acetone. Pellets were air dried. Protein pellets were then resuspended in 200µL TFE (2,2,2-trifluoroethanol) digestion buffer (11% TFE, 100mM NH4HCO3) and the pellets dissolved by sonication using a Branson probe sonifier until a homogenous suspension was formed. Samples were digested overnight at 37°C using mass spectrometry grade trypsin/Lys-C mix (enzyme/substrate of 1:50 w/w, 4µg) while mixing at 550 rpm. The samples were acidified on the next day with trifluoroacetic acid (TFA) added to a final concentration of 0.5% and centrifuged 10 minutes at 16,000g. H2O2 was added to each sample at a f.c. of 0.5% and incubated for 30 minutes at 37 °C. Solid phase extraction of peptides was performed using C18 reversed phase sorbent containing 100µL pipette tips (Piece C18 tips – Thermo Scientific) according to the manufacturer’s instructions. The samples were then vacuum-dried in a SpeedVac concentrator and re-dissolved in 20µL 2 mM TCEP (tris(2-carboxyethyl)phosphine) in water/ACN (98:2, v/v) for LC-MS/MS analysis. Samples were analyzed by LC-MS/MS using an UltiMate 3000 RSLC nano HPLC (Dionex) in-line connected to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific Inc.). Samples were separated on a 40 cm column packed in the needle (produced in-house, 75 μm I.D. × 400 mm, 1.9 μm beads C18 Reprosil-HD, Dr. Maisch) using a non-linear 150 min gradient of 2-56% solvent B’ (0.1% formic acid (FA) in water/ACN, 20/80 (v/v)) at a flow rate of 250 nL/min. This was followed by a 10 min wash reaching 99% solvent B’ and re-equilibration with solvent A (0.1% FA in water). Column temperature was kept constant at 50°C (CoControl 3.3.05, Sonation). The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 16 most abundant peaks in a given MS spectrum. The source voltage was set to 3.5 kV and the capillary temperature was 250°C. One MS1 scan (m/z 375-1500, AGC target 3E6 ions, maximum ion injection time of 45 ms) acquired at a resolution of 60,000 (at 200 m/z) was followed by up to 16 tandem MS scans (resolution 15,000 at 200 m/z) of the most intense ions fulfilling predefined selection criteria (AGC target 1E5 ions, maximum ion injection time of 60 ms, isolation window of 1.5 m/z, fixed first mass of 145 m/z, spectrum data type: centroid, under fill ratio 2%, intensity threshold 1.3E4, exclusion of unassigned, singly charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time of 12 s). The HCD collision energy was set to 32% Normalized Collision Energy and the polydimethylcyclosiloxane background ion at 445.12002 Da was used for internal calibration (lock mass).

Data Processing Protocol

Spectra identification was performed with MaxQuant (version 1.5.3.30) using the Andromeda search engine45 with FDR set at 1% on peptide and protein level. Spectra were searched against the “aTIS database” (in-silico translated ORFs of protein-coding transcripts annotated in Ensembl 82 Sep 2015) or “custom database” (in-silico translated ORFs both annotated in Ensembl 82 Sep 2015 and predicted based on HCT116 ribo-seq data presented in this article). The mass tolerance for precursor and fragment ions was set to 4.5 and 20 ppm, respectively, during the main search. Methionine oxidation to methionine-sulfoxide was set as a fixed modification. Acetylation of protein N-termini was set as a variable modification. Trypsin/P was set as enzyme allowing for 2 missed cleavages. The match between runs function was enabled and proteins were quantified by the MaxLFQ algorithm integrated in the MaxQuant software. Minimum of 2 ratio counts and only unique peptides were considered for protein quantification. In the "custom database" search, razor peptides were also considered, however the quantitative aspect of this data was not used in further analysis.

Contact

Daria Gawron, VIB Department of Medical Protein Research, University of Ghent
Kris Gevaert, VIB Medical Biotechnology Center Department of Biochemistry, Ghent University A. Baertsoenkaai 3 B9000 Ghent Belgium ( lab head )

Submission Date

15/09/2016

Publication Date

13/09/2017

Tissue

colon

Instrument

Q Exactive

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Fijalkowska D, Verbruggen S, Ndah E, Jonckheere V, Menschaert G, Van Damme P. eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs. Nucleic Acids Res. 2017 45(13):7997-8013 PubMed: 28541577