Abstract
Protein biogenesis at the endoplasmic reticulum (ER) in eukaryotic cells is monitored by a protein quality control system named ER-associated protein degradation (ERAD). While there has been substantial progress in understanding how ERAD eliminates defective polypeptides generated from erroneous folding, how cells remove nascent chains stalled in the translocon during co-translational protein insertion into the ER is unclear. Here we show that ribosome stalling during protein translocation induces the attachment of UFM1, a ubiquitin-like modifier, to two conserved lysine residues near the COOH-terminus of the 60S ribosomal subunit RPL26 (uL24) at the ER. Strikingly, RPL26 UFMylation enables the degradation of stalled nascent chains, but unlike ERAD or previously established cytosolic ribosome-associated quality control (RQC), which uses proteasome to degrade their client proteins, ribosome UFMylation promotes the targeting of a translocation-arrested ER protein to lysosomes for degradation. RPL26 UFMylation is upregulated during erythroid differentiation to cope with increased secretory flow, and compromising UFMylation impairs protein secretion, and ultimately hemoglobin production. We propose that in metazoan, co-translational protein translocation into the ER is safeguarded by a UFMylation-dependent protein quality control mechanism, which when impaired causes anemia in mice and abnormal neuronal development in humans.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
References
Christianson, J. C. & Ye, Y. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat. Struct. Mol. Biol. 21, 325–335 (2014).
Ruggiano, A., Foresti, O. & Carvalho, P. Quality control: ER-associated degradation: protein quality control and beyond. J. Cell Biol. 204, 869–879 (2014).
Needham, P. G., Guerriero, C. J. & Brodsky, J. L. Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases. Cold Spring Harb. Perspect. Biol. 11, a033928 (2019).
Klauer, A. A. & van Hoof, A. Degradation of mRNAs that lack a stop codon: a decade of nonstop progress. Wiley Interdiscip. Rev. RNA 3, 649–660 (2012).
Brandman, O. & Hegde, R. S. Ribosome-associated protein quality control. Nat. Struct. Mol. Biol. 23, 7–15 (2016).
Joazeiro, C. A. P. Ribosomal stalling during translation: providing substrates for ribosome-associated protein quality control. Annu Rev. Cell Dev. Biol. 33, 343–368 (2017).
Brandman, O. et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell 151, 1042–1054 (2012).
Juszkiewicz, S. & Hegde, R. S. Initiation of quality control during Poly(A) translation requires site-specific ribosome ubiquitination. Mol. Cell 65, 743–750 e744 (2017).
Matsuo, Y. et al. Ubiquitination of stalled ribosome triggers ribosome-associated quality control. Nat. Commun. 8, 159 (2017).
Sundaramoorthy, E. et al. ZNF598 and RACK1 regulate mammalian ribosome-associated quality control function by mediating regulatory 40S ribosomal ubiquitylation. Mol. Cell 65, 751–760 e754 (2017).
Shao, S., von der Malsburg, K. & Hegde, R. S. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation. Mol. Cell 50, 637–648 (2013).
Juszkiewicz, S. et al. ZNF598 is a quality control sensor of collided ribosomes. Mol. Cell 72, 469–481 e467 (2018).
Shao, S. & Hegde, R. S. Reconstitution of a minimal ribosome-associated ubiquitination pathway with purified factors. Mol. Cell 55, 880–890 (2014).
Bengtson, M. H. & Joazeiro, C. A. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467, 470–473 (2010).
Yonashiro, R. et al. The Rqc2/Tae2 subunit of the ribosome-associated quality control (RQC) complex marks ribosome-stalled nascent polypeptide chains for aggregation. Elife 5, e11794 (2016).
Shao, S., Brown, A., Santhanam, B. & Hegde, R. S. Structure and assembly pathway of the ribosome quality control complex. Mol. Cell 57, 433–444 (2015).
von der Malsburg, K., Shao, S. & Hegde, R. S. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon. Mol. Biol. Cell 26, 2168–2180 (2015).
Choe, Y. J. et al. Failure of RQC machinery causes protein aggregation and proteotoxic stress. Nature 531, 191–195 (2016).
Chu, J. et al. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration. Proc. Natl Acad. Sci. USA 106, 2097–2103 (2009).
Shen, P. S. et al. Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains. Science 347, 75–78 (2015).
Liakath-Ali, K. et al. An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis. Nature 556, 376–380 (2018).
Ishimura, R. et al. RNA function. Ribosome stalling induced by mutation of a CNS-specific tRNA causes neurodegeneration. Science 345, 455–459 (2014).
Komatsu, M. et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J. 23, 1977–1986 (2004).
Cai, Y. et al. UFBP1, a key component of the Ufm1 conjugation system, is essential for ufmylation-mediated regulation of erythroid development. PLoS Genet. 11, e1005643 (2015).
Tatsumi, K. et al. The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice. Nat. Commun. 2, 181 (2011).
Zhang, M. et al. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis. Cell Death Differ. 22, 1922–1934 (2015).
Nahorski, M. S. et al. Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development. Brain 141, 1934–1945 (2018).
Muona, M. et al. Biallelic variants in UBA5 link dysfunctional UFM1 ubiquitin-like modifier pathway to severe infantile-onset encephalopathy. Am. J. Hum. Genet. 99, 683–694 (2016).
Colin, E. et al. Biallelic variants in UBA5 reveal that disruption of the UFM1 cascade can result in early-onset encephalopathy. Am. J. Hum. Genet. 99, 695–703 (2016).
Duan, R. et al. UBA5 mutations cause a new form of autosomal recessive cerebellar ataxia. PLoS ONE 11, e0149039 (2016).
Yoo, H. M. et al. Modification of ASC1 by UFM1 is crucial for ERalpha transactivation and breast cancer development. Mol. Cell 56, 261–274 (2014).
Liu, J. et al. A critical role of DDRGK1 in endoplasmic reticulum homoeostasis via regulation of IRE1alpha stability. Nat. Commun. 8, 14186 (2017).
Qin, B. et al. UFL1 promotes histone H4 ufmylation and ATM activation. Nat. Commun. 10, 1242 (2019).
Simsek, D. et al. The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity. Cell 169, 1051–1065 e1018 (2017).
Walczak, C. P. et al. Ribosomal protein RPL26 is the principal target of UFMylation. Proc. Natl Acad. Sci. USA 116, 1299–1308 (2019).
Tatsumi, K. et al. A novel type of E3 ligase for the Ufm1 conjugation system. J. Biol. Chem. 285, 5417–5427 (2010).
Kang, S. H. et al. Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2. J. Biol. Chem. 282, 5256–5262 (2007).
Lemaire, K. et al. Ubiquitin fold modifier 1 (UFM1) and its target UFBP1 protect pancreatic beta cells from ER stress-induced apoptosis. PLoS ONE 6, e18517 (2011).
Garreau de Loubresse, N. et al. Structural basis for the inhibition of the eukaryotic ribosome. Nature 513, 517–522 (2014).
Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).
Pestka, S. Inhibitors of ribosome functions. Annu. Rev. Microbiol 25, 487–562 (1971).
Dimitrova, L. N., Kuroha, K., Tatematsu, T. & Inada, T. Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome. J. Biol. Chem. 284, 10343–10352 (2009).
Verma, R. et al. Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes. Nature 557, 446–451 (2018).
Rubinsztein, D. C. Cell biology: receptors for selective recycling. Nature 522, 291–292 (2015).
Wyant, G. A. et al. NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science 360, 751–758 (2018).
Andersson, L. C., Jokinen, M. & Gahmberg, C. G. Induction of erythroid differentiation in the human leukaemia cell line K562. Nature 278, 364–365 (1979).
An, X. et al. Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 123, 3466–3477 (2014).
Hu, X. et al. Ubiquitin-fold modifier 1 inhibits apoptosis by suppressing the endoplasmic reticulum stress response in Raw264.7 cells. Int. J. Mol. Med. 33, 1539–1546 (2014).
Zhang, Y., Zhang, M., Wu, J., Lei, G. & Li, H. Transcriptional regulation of the Ufm1 conjugation system in response to disturbance of the endoplasmic reticulum homeostasis and inhibition of vesicle trafficking. PLoS ONE 7, e48587 (2012).
Crowder, J. J. et al. Rkr1/Ltn1 ubiquitin ligase-mediated degradation of translationally stalled endoplasmic reticulum proteins. J. Biol. Chem. 290, 18454–18466 (2015).
Ast, T., Michaelis, S. & Schuldiner, M. The protease Ste24 clears clogged translocons. Cell 164, 103–114 (2016).
Kuroha, K., Zinoviev, A., Hellen, C. U. T. & Pestova, T. V. Release of ubiquitinated and non-ubiquitinated nascent chains from stalled mammalian ribosomal complexes by ANKZF1 and Ptrh1. Mol. Cell 72, 286–302 e288 (2018).
Zurita Rendon, O. et al. Vms1p is a release factor for the ribosome-associated quality control complex. Nat. Commun. 9, 2197 (2018).
Mills, E. W., Wangen, J., Green, R. & Ingolia, N. T. Dynamic regulation of a ribosome rescue pathway in erythroid cells and platelets. Cell Rep. 17, 1–10 (2016).
Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).
Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl Acad. Sci. USA 113, E3501–E3508 (2016).
Guydosh, N. R. & Green, R. Dom34 rescues ribosomes in 3’ untranslated regions. Cell 156, 950–962 (2014).
Belin, S. et al. Purification of ribosomes from human cell lines. in Current Protocols in Cell Biology, Ch. 3, Unit 3 40, https://doi.org/10.1002/0471143030.cb0340s49 (2010).
Fibach, E. Techniques for studying stimulation of fetal hemoglobin production in human erythroid cultures. Hemoglobin 22, 445–458 (1998).
Vaisman, B., Meyron-Holtz, E. G., Fibach, E., Krichevsky, A. M. & Konijn, A. M. Ferritin expression in maturing normal human erythroid precursors. Br. J. Haematol. 110, 394–401 (2000).
Myasnikov, A. G. et al. Structure-function insights reveal the human ribosome as a cancer target for antibiotics. Nat. Commun. 7, 12856 (2016).
Behrmann, E. et al. Structural snapshots of actively translating human ribosomes. Cell 161, 845–857 (2015).
Acknowledgements
We thank Taplin mass spectrometry facility (Harvard Medical School) for protein identification service, NHLBI genomic core for RNA sequencing, W. Chen (NIDDK, NIH) for RNA sequence analysis, T. Rapoport for Sec61β antibodies, R. Hegde for NEMF antibodies, and the members of the Ye laboratory for discussions. L.W., Y.X., L.S., H.R., Y.Y., C.T.N., and N.R.G. are supported by an intramural research program of NIDDK in the National Institutes of Health, J.W.Y. is supported by an intramural research program of NIAID in the National Institutes of Health, H.L. is supported by NIH RO1DK113409.
Author information
Authors and Affiliations
Contributions
L.W., X.Y., and Y.Y. designed the research. L.W., X.Y., L.S., and Y.Y. performed experiments. H.L. and J.W.Y. provided essential reagents and helped confirm RPL26 as a UFM1 substrate, N.R.G. contributed to the idea that ribosome stalling triggers UFMylation. H.R. and C.T.N. provides HSPCs. L.W. and Y.Y. wrote the paper and all authors participated in editing the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Supplementary information
Rights and permissions
About this article
Cite this article
Wang, L., Xu, Y., Rogers, H. et al. UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis. Cell Res 30, 5–20 (2020). https://doi.org/10.1038/s41422-019-0236-6
Received:
Accepted:
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41422-019-0236-6
This article is cited by
-
The UFL1-AKT positive feedback loop promotes breast cancer progression by enhancing lipid synthesis
Nature Communications (2026)
-
The mechanistic basis and cellular functions of UFMylation
Nature Reviews Molecular Cell Biology (2026)
-
Quality control and signaling pathways at stalled ribosomes
Experimental & Molecular Medicine (2026)
-
UFMylation in lipid metabolism disorders-associated diseases
Molecular Biology Reports (2026)
-
The many connections of UFMylation with Alzheimer’s disease: a comprehensive review
Molecular Neurodegeneration (2025)
