Peptide and protein identification
MS spectra were evaluated by Cell Signaling Technology using SEQUEST
and the GFY-Core platform (Harvard University)^38 –^40. Searches were
performed against the most recent update of the Uniprot Mus musculus
database (20190703 update) with a mass accuracy of ±50 ppm for pre-
cursor ions and 0.02 Da for productions. Cysteine carboxamidometh-
ylation was specified as a static modification (C + 57.02146374) as was
TMT labelling of lysine (K + 229.162932) and TMT labelling of N termini
(Nterm + 229.162932). Oxidation of methionine (M + 15.9949146221)
was specified as a variable modification. Results were filtered to a 1%
peptide-level false discovery rate (FDR) and further filtered to a 1%
protein level FDR. TMT quantitative results were generated in GFY-Core
based on reporter ion signal to noise values in each channel. Individual
peptide signal to noise values were summed for each protein, and rela-
tive protein abundance between channels was compared. P values were
based on a two-tailed t-test across replicates.
RiboTag assays, RNA sequencing library preparation and analysis
Frozen hippocampi from one mouse were pooled and homogenized in
1 ml buffer as described^41. After centrifugation, 4 μl anti-HA antibody
(Supplementary Table 3) was added to 800 μl cleared lysate and incu-
bated for 4 h at 4 °C. The remaining lysate was saved as input sample.
After incubation, 200 μl protein A/G magnetic beads (Thermo Fisher
Scientific) was added and incubated overnight at 4 °C with rotation.
Immunoprecipitates (IPs) were washed in high-salt buffer and RNA from
inputs and IPs extracted as described in the original protocol^42. RNA-seq
libraries were prepared from 200 ng total RNA using the NEBNext Ultra
II Directional RNA Library Prep Kit for Illumina with the NEBNext Poly(A)
mRNA Magnetic Isolation Module and NEBNext Multiplex Oligos (New
England Biolabs). Libraries were pair-end sequenced (2 × 50 bp) on
an Illumina NovaSeq Instrument. The adaptor sequence GATCGGAA-
GAGCACACG was removed from reads using Cutadapt. Reads shorter
than 25 nucleotides were discarded. Reads were aligned to ribosomal
RNA using Bowtie^43 , allowing for two mismatches and no ambiguous
mapping, and aligned reads were discarded. The remaining forward
pair reads were then aligned to the mouse transcriptome (Gencode
version 14) using Bowtie, allowing for two mismatches and no ambigu-
ous mapping^43 ,^44. Differential expression analysis was carried out using
DESeq2 version 1.26.0^45. To examine a global correlation between the
effect of ablation of p-eIF2α and learning, we calculated Pearson cor-
relations between log 2 fold change values. The Pearson correlation and
significance test were done using R. Functional enrichment analysis for
subsets of transcripts or proteins was calculated using Enricher tool
(EnrichR)^46. Enrichment was considered significant for FDR-adjusted
P values <0.1. The full dataset of the RiboTag gene-expression data is
available at the National Centre for Biotechnology Information Gene
Expression Omnibus (GEO accession number GSE152825).
Statistical analysis
Data are presented as mean ± s.e.m. The statistical tests were per-
formed using GraphPad Prism 7.00 (GraphPad Prism Software Inc.,
USA). The statistical significance of differences between two groups
was determined using two-tailed unpaired Student’s t-test with Welch’s
correction. Multiple groups were compared by one-way ANOVA or
two-way ANOVA. The statistical significance differences at different
time points were determined using a two-way repeated measure ANOVA
or mixed-effects model (REML). The post-hoc test (Tukey’s or Sidak’s,
α level of 0.05 and P < 0.05) used to compare individual groups is indi-
cated in the figure legends. All statistical comparisons are listed in
Supplementary Table 4.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.
Data availability
The full RiboTag gene-expression dataset is available at the National
Centre for Biotechnology Information Gene Expression Omnibus (GEO
accession number GSE152825). The additional relevant data that sup-
port the findings of this study are available from the corresponding
author upon reasonable request. Source data are provided with this
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Acknowledgements This study was supported by a Canada’s International Development
Research Centre (IDRC), in partnership with the Azrieli Foundation, the Canadian Institutes
of Health Research (CIHR), and the Israel Science Foundation (ISF) to K.R. and N.S. J.-C.L. is
supported by a CIHR Project grant (PJT-153311) and a Canada Research Chair in Cellular
and Molecular Neurophysiology (CRC-950-231066). R.J.K. is supported by National
Institutes of Health (NIH) Project grants (R01 DK113171, R01 DK103185, R01 CA198103 and
R01 AG062190). E.S. is supported by the Ministerio de Ciencia, Innovación y Universidades
(RTI2018-101838-J-I00) and A.Q. is supported by the European Research Council
(ERC-2014-StG-638106), Ministerio de Economía y Competitividad (SAF2017-88108-R) and
Agència de Gestió d’Ajuts Universitaris i de Recerca (2017SGR- 323). M.C.-M. is supported
by the National Institute of Neurological Disorders and Stroke grant (2R01 NS076708-06
NINDS). Support to R.S. was provided by Richard and Edith Strauss Postdoctoral
Fellowships in Medicine. We thank the CNAG-CRG for assistance with RNA sequencing;
G. S. McKnight for the RPL22-HA plasmid; members of the Sonenberg laboratory,
specifically I. Harvey, A. Lafrance, A. Sylvestre, E. Migon and S. Murthy, as well as
I. Laplante, H. Hall, M. Anadolu and K. Gamache for support with animals and resources;
and S. Tahmasebi for critical reading of the manuscript.
Author contributions V.S., A. Khoutorsky, J.-C.L., K.R. and N.S. conceived the project,
designed experiments and supervised the research. V.S., R.S., T.-Y.H., D.L. and N.C. designed
and set up mouse breeding. V.S., R.S., T.-Y.H., D.L., N.C. and V.T.T. performed the stereotaxic
surgery, cannula implantation and microinjection. V.S., R.S., T.-Y.H. and D.L. carried out
iSUnSET and FUNCAT. A. Khlaifia, A.A. and M.J.E. carried out whole-cell recording and
analysis. V.S., A. Khlaifia, A.A., M.J.E. and F.S. carried out field potential recordings and
analysis. V.S., R.S., T.-Y.H., D.L. and N.C. carried out behaviour tests, immunohistochemistry
and image analysis. V.S., R.S., T.-Y.H., D.L., M.L., M.P.S., A.J.N., K.A. and A.P.P. carried out
sample preparation and LC–MS/MS analysis. V.S., E.S., M.L., S.J.K., R.S., T.-Y.H., D.L. and P.W.
carried out RiboTag assays, RNA sequencing library preparation and analysis. V.S., R.S.,
A. Khlaifia, T.-Y.H., D.L., A.A., M.L., N.C., S.J.K., E.S., P.V.B., M.C.-M., A. Khoutorsky, J.-C.L., K.R.
and N.S. wrote the manuscript. S.G.-B.-A., A.C.C., K.N., A.Q. and R.J.K. supported the
experiments. All authors reviewed the manuscript and discussed the work.Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2805-8.
Correspondence and requests for materials should be addressed to V.S., K.R. or N.S.
Peer review information Nature thanks the anonymous reviewers for their contribution to the
peer review of this work.
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