Functional Characterization of a Putative RNA Demethylase ALKBH6 in Arabidopsis Growth and Abiotic Stress Responses

Abstract

RNA methylation and demethylation, which is mediated by RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively, are emerging as a key regulatory process in plant development and stress responses. Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases, the function of most ALKBHs is yet to be determined. The Arabidopsis thaliana genome contains thirteen genes encoding ALKBH proteins, the functions of which are largely unknown. In this study, we characterized the function of a potential eraser protein, ALKBH6 (At4g20350), during seed germination and seedling growth in Arabidopsis under abiotic stresses. The seeds of T-DNA insertion alkbh6 knockdown mutants germinated faster than the wild-type seeds under cold, salt, or abscisic acid (ABA) treatment conditions but not under dehydration stress conditions. Although no differences in seedling and root growth were observed between the alkbh6 mutant and wild-type under normal conditions, the alkbh6 mutant showed a much lower survival rate than the wild-type under salt, drought, or heat stress. Cotyledon greening of the alkbh6 mutants was much higher than that of the wild-type upon ABA application. Moreover, the transcript levels of ABA signaling-related genes, including ABI3 and ABI4, were down-regulated in the alkbh6 mutant compared to wild-type plants. Importantly, the ALKBH6 protein had an ability to bind to both m6A-labeled and m5C-labeled RNAs. Collectively, these results indicate that the potential eraser ALKBH6 plays important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses.

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus

“…The Arabidopsis genome encodes 14 homologs of Alk B family of “eraser” proteins ( at ALKBH1A-D, at ALKBH2, at ALKBH6, at ALKBH8A-B, at ALKBH9A-C, and at ALKBH10A-B) ( Mielecki et al, 2012 ; Kawai et al, 2014 ), of which, at ALKBH9B and at ALKBH10B, have been shown to present m 6 A demethylase activity in vitro and m 6 A-related functions in vivo ( Duan et al, 2017 ; Martínez-Pérez et al, 2017 ). Recently the potential eraser at ALKBH6 has been shown to play important roles in seed germination, seedling growth, and survival of Arabidopsis under abiotic stresses ( Huong et al, 2020 ). Interestingly, at ALKBH9B is the only m 6 A demethylase that is located exclusively in the cytoplasm ( Mielecki et al, 2012 ), forming granules that colocalize with SGS3 (a component of siRNA bodies) and in some cases, associates with DCP1 (P bodies) ( Ingelfinger et al, 2002 ; Martínez de Alba et al, 2015 ; Martínez-Pérez et al, 2017 ).…”

5-methylcytosine modification byPlasmodiumNSUN2 stabilizes mRNA and mediates the development of gametocytes

“…In another example, the DEAD/DEAH helicase (PY17X_0313200), an AP2-Ginduced transcript involved in male gametocyte development in P. berghei (5), was found to be m 5 C methylated in both asexual and sexual stages of P. yoelii. Similarly, data from P. falciparum showed stage-specific m 5 C methylation of three transcripts that possibly regulate sexual commitment and gametocytogenesis (2,3,23), including genes whose disruption is known to lead to greatly reduced gametocyte numbers (GIG, AP2-G2, Pfs16) (2, 3) (SI Appendix, Fig. S3A).…”

“…ALYREF is an adaptor that regulates the transport of m 5 C-modified transcripts from the nucleus to the cytoplasm (14), whereas Ybx1 promotes the stability of its target mRNAs in an m 5 C-dependent manner (21). A potential m C demethylase, ALKBH (an AlkB homolog), was reported more recently from Arabidopsis and is attracting study for its possible role as an m 5 C "eraser" (22,23).…”

RNA N6-Methyladenosine Responds to Low-Temperature Stress in Tomato Anthers

“…Growing evidence suggests that diverse abiotic stresses can change transcript m 6 A methylation levels (Hu et al, 2019;Huong et al, 2020;Liu et al, 2020). Moreover, m 6 A levels usually negatively regulate gene expression by influencing mRNA stability and the subsequent protein synthesis process (Shen et al, 2016;Duan et al, 2017;Zhao et al, 2017).…”

“…In Arabidopsis, changed m 6 A deposition affects RNA secondary structure under salt stress, resulting in increased stability of mRNA transcripts of abiotic stress response genes (Kramer et al, 2020). Additionally, ALKBH6 (eraser) mutation results in increased salt, drought, and heat stress sensitivity during seed germination (Huong et al, 2020).…”