Abstract
Stress granules are dynamic structures that assemble in response to various forms of stress, such as heat shock or oxidative stress, among others. We had previously shown that the lysine deacetylase HDAC6 is required for the formation of stress granules, but the substrate important for this function was not known. We recently found that the RNA helicase DDX3X is a novel HDAC6 substrate, which is critical for the formation of stress granules. Through a series of detailed experiments, we showed that, upon stress, DDX3X becomes acetylated in an intrinsically disordered region; this alters its propensity to undergo phase separation and inhibits growth of the stress granules. HDAC6, by deacetylating DDX3X, allows maturation of the stress granules. This work identified acetylation of an RNA helicase as a critical regulator of the stress response. Here, we present methods to analyze the acetylation of DDX3X; these methods can be easily adapted to study the acetylation of other helicases, or other proteins.
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Allfrey VG, Faulkner R, Mirsky AE (1964) Acetylation and methylation of histones and their possible role in the regulation of Rna synthesis. Proc Natl Acad Sci U S A 51:786–794
Verdin E, Ott M (2015) 50 years of protein acetylation: from gene regulation to epigenetics, metabolism and beyond. Nat Rev Mol Cell Biol 16:258–264
Li L, Yang XJ (2015) Tubulin acetylation: responsible enzymes, biological functions and human diseases. Cell Mol Life Sci 72:4237–4255
Choudhary C, Kumar C, Gnad F et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834–840
Choudhary C, Weinert BT, Nishida Y et al (2014) The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15:536–550
Matthias P, Yoshida M, Khochbin S (2008) HDAC6 a new cellular stress surveillance factor. Cell Cycle 7:7–10
Miyake Y, Keusch JJ, Wang L et al (2016) Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat Chem Biol 12:748–754
Kawaguchi Y, Kovacs JJ, McLaurin A et al (2003) The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell 115:727–738
Boyault C, Zhang Y, Fritah S et al (2007) HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev 21:2172–2181
Kwon S, Zhang Y, Matthias P (2007) The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev 21:3381–3394
Choi SJ, Lee HC, Kim JH et al (2016) HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I. EMBO J 35:429–442
Banerjee I, Miyake Y, Nobs SP et al (2014) Influenza a virus uses the aggresome processing machinery for host cell entry. Science 346:473–477
Saito M, Hess D, Eglinger J et al (2019) Acetylation of intrinsically disordered regions regulates phase separation. Nat Chem Biol 15:51–61
Sharma D, Jankowsky E (2014) The Ded1/DDX3 subfamily of DEAD-box RNA helicases. Crit Rev Biochem Mol Biol 49:343–360
Bol GM, Xie M, Raman V (2015) DDX3, a potential target for cancer treatment. Mol Cancer 14:188
Samir P, Kesavardhana S, Patmore DM et al (2019) DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome. Nature 573:590–594
Zhang Y, Kwon S, Yamaguchi T et al (2008) Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol Cell Biol 28:1688–1701
Scholz C, Weinert BT, Wagner SA et al (2015) Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat Biotechnol 33:415–423
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Saito, M., Iestamantavicius, V., Hess, D., Matthias, P. (2021). Monitoring Acetylation of the RNA Helicase DDX3X, a Protein Critical for Formation of Stress Granules. In: Boudvillain, M. (eds) RNA Remodeling Proteins. Methods in Molecular Biology, vol 2209. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0935-4_14
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DOI: https://doi.org/10.1007/978-1-0716-0935-4_14
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