Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing

Nature Cell Biology volume 25, pages 754–764 (2023)Cite this article

Subjects

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is an essential hub that integrates nutrient signals and coordinates metabolism to control cell growth. Amino acid signals are detected by sensor proteins and relayed to the GATOR2 and GATOR1 complexes to control mTORC1 activity. Here we perform genome-wide CRISPR/Cas9 screens, coupled with an assay for mTORC1 activity based on fluorescence-activated cell sorting analysis of pS6, to identify potential regulators of mTORC1-dependent amino acid sensing. We then focus on interleukin enhancer binding factor 3 (ILF3), one of the candidate genes from the screen. ILF3 tethers the GATOR complexes to lysosomes to control mTORC1. Adding a lysosome-targeting sequence to the GATOR2 component WDR24 bypasses the requirement for ILF3 to modulate amino-acid-dependent mTORC1 signalling. ILF3 plays an evolutionarily conserved role in human and mouse cells, and in worms to regulate the mTORC1 pathway, control autophagy activity and modulate the ageing process.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Genome-wide CRISPR screens to identify modulators of mTORC1-dependent amino acid sensing.
Fig. 2: ILF3 functions upstream of the Rag GTPases and downstream of the GATOR complexes to regulate mTORC1 activity.
Fig. 3: ILF3 interacts with GATOR1 and GATOR2 in an amino-acid-insensitive manner.
Fig. 4: ILF3 is required for the lysosomal localization of GATOR1 and GATOR2.
Fig. 5: The lysosomal localization of ILF3i1 is independent of amino acids and autophagy.
Fig. 6: ILF3 plays an evolutionarily conserved role in mouse cells to regulate mTORC1 signalling.
Fig. 7: ILF3 plays a conserved role in C. elegans to regulate mTORC1 and ageing.

Similar content being viewed by others

Data availability

CRISPR screen data are provided in Supplementary Tables 1–3 in Excel format. Source data are provided with this paper. Data from the GTEx database used in Extended Data Fig. 2c are accessible through its official website (https://www.gtexportal.org). All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sancak, Y. et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T. P. & Guan, K. L. Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bar-Peled, L. et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chantranupong, L. et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell 165, 153–164 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wolfson, R. L. et al. Sestrin2 is a leucine sensor for the mTORC1 pathway. Science 351, 43–48 (2016).

    Article  CAS  PubMed  Google Scholar 

  7. Chen, J. et al. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature 596, 281–284 (2021).

    Article  CAS  PubMed  Google Scholar 

  8. Gu, X. et al. SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 358, 813–818 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dokudovskaya, S. et al. A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Mol. Cell Proteom. 10, M110.006478 (2011).

    Article  Google Scholar 

  10. Wu, X. & Tu, B. P. Selective regulation of autophagy by the Iml1-Npr2-Npr3 complex in the absence of nitrogen starvation. Mol. Biol. Cell 22, 4124–4133 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Neklesa, T. K. & Davis, R. W. A genome-wide screen for regulators of TORC1 in response to amino acid starvation reveals a conserved Npr2/3 complex. PLoS Genet. 5, e1000515 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Peng, M., Yin, N. & Li, M. O. SZT2 dictates GATOR control of mTORC1 signalling. Nature 543, 433–437 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wolfson, R. L. et al. KICSTOR recruits GATOR1 to the lysosome and is necessary for nutrients to regulate mTORC1. Nature 543, 438–442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Loissell-Baltazar, Y. A. & Dokudovskaya, S. SEA and GATOR 10 years later. Cells 10, 2689 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 169, 361–371 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Valvezan, A. J. & Manning, B. D. Molecular logic of mTORC1 signalling as a metabolic rheostat. Nat. Metab. 1, 321–333 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Jewell, J. L. et al. Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science 347, 194–198 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kang, J. et al. Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines. Nat. Biotechnol. 34, 70–77 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. De Zan, E. et al. Quantitative genetic screening reveals a Ragulator-FLCN feedback loop that regulates the mTORC1 pathway. Sci. Signal 13, eaba5665 (2020).

    Article  PubMed  Google Scholar 

  20. Condon, K. J. et al. Genome-wide CRISPR screens reveal multitiered mechanisms through which mTORC1 senses mitochondrial dysfunction. Proc. Natl Acad. Sci. USA 118, e2022120118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. He, X. D. et al. Sensing and transmitting intracellular amino acid signals through reversible lysine aminoacylations. Cell Metab. 27, 151–166 (2018).

    Article  CAS  PubMed  Google Scholar 

  22. Han, J. M. et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149, 410–424 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Kim, S. H. et al. Mitochondrial threonyl-tRNA synthetase TARS2 is required for threonine-sensitive mTORC1 activation. Mol. Cell 81, 398–407 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xu, Y. et al. A bacterial effector reveals the V-ATPase-ATG16L1 axis that initiates xenophagy. Cell 178, 552–566 (2019).

    Article  CAS  PubMed  Google Scholar 

  26. Li, X. et al. Coordinated circRNA biogenesis and function with NF90/NF110 in viral infection. Mol. Cell 67, 214–227 (2017).

    Article  CAS  PubMed  Google Scholar 

  27. Harashima, A., Guettouche, T. & Barber, G. N. Phosphorylation of the NFAR proteins by the dsRNA-dependent protein kinase PKR constitutes a novel mechanism of translational regulation and cellular defense. Genes Dev. 24, 2640–2653 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pfeifer, I. et al. NFAR-1 and -2 modulate translation and are required for efficient host defense. Proc. Natl Acad. Sci. USA 105, 4173–4178 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Guan, D. et al. Nuclear factor 45 (NF45) is a regulatory subunit of complexes with NF90/110 involved in mitotic control. Mol. Cell. Biol. 28, 4629–4641 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Peng, M., Yin, N. & Li, M. O. Sestrins function as guanine nucleotide dissociation inhibitors for Rag GTPases to control mTORC1 signaling. Cell 159, 122–133 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ye, J., Jin, H., Pankov, A., Song, J. S. & Blelloch, R. NF45 and NF90/NF110 coordinately regulate ESC pluripotency and differentiation. RNA 23, 1270–1284 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Abu-Remaileh, M. et al. Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes. Science 358, 807–813 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Menon, S. et al. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1. Autophagy 17, 1–382 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Zhu, H. et al. Metabolomic profiling of single enlarged lysosomes. Nat. Methods 18, 788–798 (2021).

    Article  CAS  PubMed  Google Scholar 

  37. Zhang, H. et al. Guidelines for monitoring autophagy in Caenorhabditis elegans. Autophagy 11, 9–27 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Panowski, S. H., Wolff, S., Aguilaniu, H., Durieux, J. & Dillin, A. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature 447, 550–555 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Greer, E. L. et al. An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans. Curr. Biol. 17, 1646–1656 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Heintz, C. et al. Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans. Nature 541, 102–106 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Chen, J. et al. KLHL22 activates amino-acid-dependent mTORC1 signalling to promote tumorigenesis and ageing. Nature 557, 585–589 (2018).

    Article  CAS  PubMed  Google Scholar 

  42. Liu, G. Y. & Sabatini, D. M. mTOR at the nexus of nutrition, growth, ageing and disease. Nat. Rev. Mol. Cell Biol. 21, 183–203 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Long, L. et al. CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity. Nature 600, 308–313 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Joung, J. et al. Genome-scale CRISPR–Cas9 knockout and transcriptional activation screening. Nat. Protoc. 12, 828–863 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhou, P. et al. Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose. Nature 561, 122–126 (2018).

    Article  CAS  PubMed  Google Scholar 

  46. Yan, G. et al. KAT7-mediated CANX (calnexin) crotonylation regulates leucine-stimulated MTORC1 activity. Autophagy 18, 2799–2816 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wyant, G. A. et al. NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science 360, 751–758 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wolfson, R. L. & Sabatini, D. M. The dawn of the age of amino acid sensors for the mTORC1 pathway. Cell Metab. 26, 301–309 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank H. He (National Institute of Biological Sciences) for assistance with CRISPR screening. We thank L. Chen for providing the shRNA plasmid targeting ILF3 isoform 1, and H. Zhang for providing the GFP::LGG-1 worm strain. The ATG5-knockout HEK293T cell line was a gift from J. Cui. Y.L. was supported by grants from the National Key Research and Development Program of China (grant no. 2022YFA0806502), the National Natural Science Foundation of China (grant nos. 92157301, 31925012 and 92254305) and the HHMI International Research Scholar Program (grant no. 55008739). Y.L. acknowledges support from the Tencent Foundation through the XPLORER PRIZE. G.Y. was supported by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.

Author information

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China

    Guokai Yan, Wen Li & Ying Liu

  2. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Guokai Yan, Jinxin Yang, Wen Li & Ying Liu

  3. PKU-Tsinghua-NIBS Graduate Program, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Ao Guo & Jialiang Guan

Authors
  1. Guokai Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinxin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ao Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jialiang Guan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.Y. and Y.L. conceived the study and designed the experiments. G.Y., J.Y., W.L. and A.G. performed the experiments. J.G. performed the bioinformatic analysis. G.Y. and Y.L. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Ying Liu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks Constantinos Demetriades and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling editor: Melina Casadio, in collaboration with the Nature Cell Biology team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genome-wide CRISPR/Cas9 knockout screening to identify potential regulators of mTORC1-dependent amino acid sensing.

a, HEK293T cells were starved of amino acids for different times as indicated, or starved for 2 hours and re-stimulated with amino acids for different times. Cell lysates were immunoblotted to detect the (phosphorylated) level of the indicated proteins. b, HEK293T cells were cultured with fresh complete medium, or medium lacking amino acids for 2 hours followed with or without amino acid re-stimulation for 1 hour. Cells were then immunostained with an antibody against pS6 (green), co-stained with DAPI (blue) for DNA content, and observed with a laser scanning confocal microscope. Scale bar, 100 μm. c, HEK293T cells were cultured with fresh complete medium, or medium lacking amino acids for 2 hours with or without amino acid re-stimulation for 1 hour. Cells were then immunostained with an antibody against pS6 (Alexa Fluor® 488 conjugate) and subjected to FACS-based analysis. d, The list of known regulators of mTORC1-dependent amino acid sensing identified by our CRISPR screen. e, Gene ontology (GO) analysis of the candidate genes. The top 15 enriched biological processes are shown. f, Protein-protein interaction analysis of the candidate genes. Only clustered genes are shown. Blue and red circles represent genes from the screen groups -AAs and +AAs, respectively. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 ILF3 functions specifically in the amino acid-regulated mTORC1 pathway.

a, HEK293T cells stably expressing the indicated sgRNAs were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. b, Clones were grown from single HEK293T cells expressing control (Ctrl) or ILF3 shRNAs. The selected single-cell clones were treated as in a, and cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. c, ILF3 isoforms 1 and 2 are widely expressed in most human tissues. Data are from GTEx. d, Cells expressing Ctrl or ILF3i1 sgRNAs, with or without re-introduction of ILF3i1 cDNA, were treated as in a, immunostained with an antibody against pS6 (red), co-stained with DAPI (blue) for DNA content and observed with a laser scanning confocal microscope. Scale bar, 10 μm. n = 8 biologically independent samples. Data are mean ± s.d. e, HEK293E cells expressing Ctrl or ILF3 shRNAs were starved of serum or amino acids for 1 hour, or starved and then re-stimulated with serum (10%) or amino acids for the indicated times. Cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. f,g, HEK293T cells were transfected with S6K1 cDNA combined with the indicated cDNAs, treated as in a, and immunoblotted to detect the indicated proteins and phospho-proteins. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 ILF2 interacts with ILF3 and acts as a negative regulator of amino acid-stimulated mTORC1 activity.

a, HEK293T cells transfected with the indicated cDNAs were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cell lysates were immunoprecipitated with FLAG beads. The whole-cell lysates (WCLs) and IPs were immunoblotted to detect the indicated proteins and phospho-proteins. b, HEK293T cells stably expressing Flag-tagged Metap2 or ILF3i1 were treated as in a, and then subjected to immunoprecipitation with FLAG beads. The WCLs and IPs were immunoblotted to detect the indicated proteins and phospho-proteins. c, Cells expressing Ctrl or ILF3 shRNAs were infected with lentivirus to co-express sgRNAs targeting GFP or ILF3. Cells were then treated as in a, and immunoblotted to detect the indicated proteins and phospho-proteins. d, HEK293T cells were transfected with S6K1 cDNA combined with the indicated cDNAs, treated as in a, and immunoblotted to detect the indicated proteins and phospho-proteins. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 ILF3 functions upstream of the Rag GTPases and downstream of the GATOR complexes to regulate mTORC1 activity.

a, HEK293T cells expressing Ctrl or ILF3i1 sgRNAs were transfected with S6K1 cDNA combined with the indicated cDNAs, starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min, and immunoblotted to detect the indicated proteins and phospho-proteins. b, HEK293T cells stably expressing 3×HA-tagged TMEM192, with co-expression of Ctrl or ILF3i1 sgRNAs, were treated as in a, and then subjected to an immunoprecipitation-based lysosome capture process. Whole-cell lysates (WCLs) and immunoprecipitated lysosome fractions (LysoIPs) were immunoblotted to detect the indicated proteins and phospho-proteins. c,d, HEK293T cells expressing Ctrl, ILF3i1 (c), or ILF2 (d) sgRNAs were transfected with 2 ng S6K1 cDNA combined with Flag-tagged Metap2 (100 ng) or DEPDC5 (10, 30, 100 ng) plasmids. Cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. e,f, HEK293T cells expressing Ctrl or ILF3 shRNAs were transfected with S6K1 cDNA combined with Flag-tagged Metap2, SESN2 (e), CASTOR1 (e) or SAMTOR (f) cDNAs, treated as in a, and cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 5 Domain mapping of the interaction of ILF3i1 and WDR24/WDR59.

a, Immunoblotting analysis of cell lines expressing near endogenous levels of GFP-tagged ILF3i1 or ILF3i2. b, HEK293T cells transfected with the indicated domains of ILF3i1 were subjected to immunoprecipitation with FLAG beads. The WCLs and IPs were immunoblotted to detect the indicated proteins. c,d, HEK293T cells transfected with the ILF3i1 CTD and different domains of WDR59 (c) or WDR24 (d) were subjected to immunoprecipitation with HA beads. The WCLs and IPs were immunoblotted to detect the indicated proteins. e, HEK293T cells expressing shILF3 and the indicated domains of ILF3i1 were starved of amino acids for 1 hr. Cell lysates were immunoblotted to detect the indicated proteins and phospho-proteins. f, HEK293T cells transfected with the indicated cDNAs were subjected to immunoprecipitation with anti-HA or anti-FLAG beads. The WCLs and IPs were immunoblotted to detect the indicated proteins. g, HEK293T cells stably expressing Flag-tagged Metap2 or ILF2 were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min, and then subjected to immunoprecipitation with anti-FLAG beads. The WCLs and IPs were immunoblotted to detect the indicated proteins. Unprocessed blots are available in source data.

Source data

Extended Data Fig. 6 ILF3 controls the lysosomal localization of GATOR1 and GATOR2 to regulate mTORC1 activity.

a, HEK293T cells expressing Ctrl or ILF3 shRNAs were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cells were then immunostained with antibodies against WDR59 (red) and LAMP2 (green), co-stained with DAPI (blue) for DNA content, and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. b, HEK293T cells stably expressing GFP-tagged WDR24, with co-expression of Ctrl or ILF3 shRNAs, were treated as in a. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. c, HEK293T cells expressing endogenous HA-DEPDC5, with co-expression of Ctrl or ILF3 shRNAs, were treated as in a, immunostained with antibodies against HA (red) and LAMP2 (green), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. d-f, HEK293T cells stably expressing GFP-tagged NRPL2 (d), C12orf66 (e) or ITFG2 (f), with co-expression of Ctrl or ILF3 shRNAs, were treated as in a. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. g, Quantification of the colocalization of WDR59 (a), GFP-WDR24 (b), HA-DEPDC5 (c), GFP-NPRL2 (d), GFP-C12orf66 (e) or GFP-ITFG2 (f) with LAMP2 using Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. ****P < 0.0001, n.s., not significant (unpaired two-sided Student’s t-test). Source numerical data are available in source data.

Source data

Extended Data Fig. 7 Characterization of the lysosomal localization of GATOR1, GATOR2, KICSTOR, and ILF3i1.

a, Wild-type, NPRL3 KO, or SZT2 KO HEK293T cells stably expressing GFP-WDR24 were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. b, Wild-type, MIOS KO, or NPRL3 KO HEK293T cells stably expressing GFP-C12orf66 were treated as in a, immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. c, Quantification of the colocalization of GFP-WDR24 (a) or GFP-C12orf66 (b) with LAMP2 using Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. n.s., not significant (unpaired two-sided Student’s t-test). d, Wild-type, MIOS KO, NPRL3 KO, or SZT2 KO HEK293T cells stably expressing 3×HA-tagged TMEM192 were subjected to an immunoprecipitation-based lysosome capture process. The WCLs and immunoprecipitated lysosome fractions (LysoIPs) were immunoblotted to detect the indicated proteins. Quantification was performed with ImageJ. Data are mean ± s.d. (n = 3 biologically independent experiments). Exact P values are shown in the graph (unpaired two-sided Student’s t-test). Note that all blots in the sgMIOS group were normalized with their corresponding wild type group. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 8 Characterization of the lysosomal localization of ILF3.

a, HEK293T cells stably expressing near endogenous levels of GFP-tagged ILF3i2 were starved of amino acids for 50 min, or starved and then re-stimulated with amino acids for 10 min. Cells were immunostained with an antibody against LAMP2 (red), co-stained with DAPI (blue), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. Quantification of the colocalization of cytoplasmic GFP-ILF3i2 with LAMP2 was performed with Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. n.s., not significant (unpaired two-sided Student’s t-test). b, Wild-type, LAMTOR1 KO, or RagA/B DKO HEK293T cells stably expressing 3×HA-tagged TMEM192 were subjected to an immunoprecipitation-based lysosome capture process. The WCLs and immunoprecipitated lysosome fractions (LysoIPs) were immunoblotted to detect the indicated proteins. c, Wild-type, LAMTOR1 KO, or RagA/B DKO HEK293T cells stably expressing GFP-ILF3i1 were treated as in a, immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. Quantification of the colocalization of cytoplasmic GFP-ILF3i1 with LAMP2 was performed with Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. n.s., not significant (unpaired two-sided Student’s t-test). d, Schematic depiction of the mechanism by which ILF3 regulates amino acid-dependent mTORC1 signalling. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 9 ILF3 plays an evolutionarily conserved role in regulating mTORC1 and autophagy.

a, Summary of the evolutionary conservation of ILF3 and ILF2, as well as GATOR1, GATOR2 and KICSTOR components, in model organisms. b, Quantification of the colocalization between mTOR and TMEM192-EGFP in Fig. 6b using Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. ****P < 0.0001 (unpaired two-sided Student’s t-test). c, HEK293T cells expressing Ctrl or ILF3 shRNAs were starved of amino acids for the indicated times. Cells were subjected to lysis for immunoblotting analysis of the indicated proteins and phospho-proteins. d, HEK293T cells expressing Ctrl or ILF3 shRNAs were starved of amino acids for the indicated times. Cells were subjected to Q-PCR analysis of SQSTM1 mRNA abundance. n = 3 biologically independent experiments. Data are mean ± s.d. n.s., not significant (unpaired two-sided Student’s t-test). Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 10 The gating strategy for FACS.

a, b, For FACS analysis of the -AAs group (a) and the +AAs group (b), the first gating was performed using FSC-A and SSC-A, the second gating was performed using FSC-H and FSC-W, then the last gating was performed using fluorescence intensity.

Supplementary information

Reporting Summary

Peer Review File

Supplementary Table

Supplementary Table 1. List of the normalized abundance of all the sgRNAs from the screens. Supplementary Table 2. List of the enriched sgRNAs from the screens of group −AAs. Supplementary Table 3. List of the enriched sgRNAs from the screens of group +AAs.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Unprocessed western blots.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 4

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 9

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, G., Yang, J., Li, W. et al. Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing. Nat Cell Biol 25, 754–764 (2023). https://doi.org/10.1038/s41556-023-01123-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-023-01123-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology