ALKBH3 Partner ASCC3 Mediates P-Body Formation and Selective
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Wollen et al. J Transl Med (2021) 19:287 https://doi.org/10.1186/s12967-021-02948-6 Journal of Translational Medicine RESEARCH Open Access ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA Kristian Lied Wollen1,2, Lars Hagen1,2,3, Cathrine B. Vågbø1,2,3, Renana Rabe1,2, Tobias S. Iveland1,2, Per Arne Aas1,2, Animesh Sharma1,2,3, Bjørnar Sporsheim1,4, Hilde O. Erlandsen1, Vuk Palibrk1, Magnar Bjørås1, Davi M. Fonseca1,2,3, Nima Mosammaparast5 and Geir Slupphaug1,2,3* Abstract Background: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifcations and whether cells distinguish aberrant from canonical methylations. Methods: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. Results: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous meth- ylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the afected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We fnd that ASCC3-defcient cells display delayed removal of MMS- induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. Conclusions: Our fndings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our fndings constitute frst evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated efects of chemical alkylators commonly used in the clinic. Keywords: Alkylating agents, ALKBH3, ASCC3, Epitranscriptome, 1-Methyladenosine, 3-Methylcytosine, 7-Methylguanosine, No-go decay, P-bodies, Ribosome quality control Background Enzymatic modifcation of nitrogen bases in mRNA *Correspondence: [email protected] regulates processing, transport, lifetime and transla- 1 Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, NTNU, 7491 Trondheim, Norway tion of mRNA molecules. In turn, this infuences cell Full list of author information is available at the end of the article diferentiation, stress responses, immunity, cognition © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wollen et al. J Transl Med (2021) 19:287 Page 2 of 22 and cancer development ([1–3] and references therein). from ALKBH3 knockout cells [15], whereas hundreds of To date, 11 endogenous base modifcations have been sites have been identifed in wild type cells [36]. Whether described in mammalian mRNA. Tese are 7-methyl- ALKBH3 shares a similar function in mRNA demeth- guanosine (m7G, residing in the mRNA cap structure ylation of endogenous m 3C [13], remains unknown. [4] as well as internally [5–7]), pseudouridine (Ψ) [8], Te YTH-domain containing proteins YTHDF1-3 and inosine (I) [9], N4-acetylcytidine (ac4C) [10], 5-methyl- YTHDC1, but not YTHDC2, have been reported to be cytidine (m5C) [11], 5-hydroxymethylcytidine (hm 5C) m1A reader proteins [26, 37]. Presently, no readers are [12], 3-methylcytidine (m3C) [13], 1-methyladeno- known for m3C or internal m7G in mRNA and no stud- sine (m1A) [14, 15], 6-methyladenosine (m6A) [16, 17] ies have attempted to monitor alterations in the human and its derivatives 6-hydroxymethyladenosine (hm 6A) mRNA binding proteome after treatment with methylat- and 6-formyladenosine (f6A) [18]. Moreover, in silico ing agents. analysis recently suggested a large number of potential To begin addressing these issues, we separately quanti- 5-methyluridine (m5U) modifcation sites in mRNA [19]. fed endogenous and MMS-induced methylbases in HeLa Additional methylations at the ribose moieties further mRNA after MMS treatment and monitored concomi- contribute to this complexity [2, 20]. Enzyme “writers” tant alterations in the mRNA binding proteome. Imme- that are able to establish each of the base modifcations diately after MMS treatment, we observed markedly have been described, and enzymes that catalyze their increased levels of m1A, m3C and m7G in mRNA. Tis removal (“erasers”) have been described for the methyl- was accompanied by reduced binding of protein mem- ated bases except internal m7G. Proteins that specifcally bers of the ribosomal 40S subunit, whereas binding of bind to and dictate the functional outcomes (“readers”) of 60S proteins was essentially unafected. Te selective 40S the base modifcations in mRNA are much less charac- loss could be explained by ribosomal collisions at MMS- terized, and are primarily confned to m6A, m1A and the induced methylbases or secondary structures, thereby m7G mRNA cap [21–27]. activating the ribosomal quality control (RQC) pathway Notably, at least three of the endogenous mRNA modi- (reviewed in [38]), GIGYF2/4EHP-dependent sequestra- fcations, m1A and m3C and m7G, may also be introduced tion of the mRNA cap and blocked recruitment of 43S in nucleic acids by direct non-enzymatic methylation preinitiation complexes (PICs) to afected mRNAs [39, by endogenous and environmental methylating agents, 40]. including tobacco-specifc nitrosamines and agents fre- ASCC3 was among the very few proteins that increased quently used as cytostatic drugs in cancer treatment [28, binding to mRNA after MMS treatment. ASCC3 is a 29]. Although chemical methylation of human mRNA DNA helicase that associates with ALKBH3 to facili- has previously not been specifcally addressed, treatment tate unwinding and efcient demethylation of m1A and of total RNA from diferent species with methyl meth- m3C in DNA [41, 42]. In addition, it was recently identi- anesulfonate (MMS) or 1-methyl-nitrosourea (MNU) fed to bind stalled ribosomes as part of the RQC trigger resulted in prominent formation of m7G as well as sev- (RQT) complex that facilitates ribosomal splitting [43]. eral fold higher relative levels of m 1A and m3C in total Our results suggest that cells exploit these properties of RNA than in DNA [28]. m1A, m3C and m7G all introduce ASCC3 to allow ALKBH3-mediated demethylation of a positive charge in addition to the methyl group. Tis aberrant m1A and m3C in the coding region of mRNA, can dramatically afect RNA–protein interactions and whereas canonical methylbases remain unafected. In secondary structures through electrostatic efects, and addition, ASCC3 has a role in P-body formation, poten- may interfere with mRNA–tRNA interactions [29–32]. tially by promoting ribosomal detachment from mRNAs A crucial question then is whether these aberrant meth- harboring difcult-to-repair lesions and assembly of such ylations are recognized and processed diferentially com- transcripts in P-bodies for further repair or degradation. pared to their endogenous counterparts. We previously Although the molecular details of such a mechanism demonstrated that the human AlkB-homolog ALKBH3 remain to be elucidated, our results provide frst evi- is able to demethylate m1A and m3C in RNA as well as dence of selective removal of aberrant methylbases from in DNA with single-strand substrate preference [33, 34]. the human mRNA pool and may have signifcant impli- Another member of the same family, ALKBH1, may con- cations to modulate the efects of cytotoxic alkylating tribute to demethylation of certain structured m1A sites drugs. in mRNA [35]. ALKBH3 is able to restore RNA function by reactivating chemically methylated RNA bacterio- Methods phages and tRNA and has been shown to act as an eraser Chemicals, plasmids and antibodies of endogenous m 1A in mRNA in vivo [15, 33]. Tousands Complete protease inhibitor cocktail was from Roche of reversible m 1A sites have been identifed in mRNA Inc. Dulbecco’s modifed Eagle medium (DMEM) minus Wollen et al. J Transl Med (2021) 19:287 Page 3 of 22 l-arginine and l-lysine, dialyzed fetal calf serum (dFCS) of l-arginine (and l-lysine) in the growth media as pre- and trypsin were from Termo Scientifc.