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

Abstract

N6-methyladenosine (m6A) modification is a dynamically regulated RNA modification that impacts many cellular processes and pathways. This epitranscriptomic methylation relies on the participation of RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively. We previously demonstrated that the Arabidopsis thaliana protein atALKBH9B showed m6A-demethylase activity and interacted with the coat protein (CP) of alfalfa mosaic virus (AMV), causing a profound impact on the viral infection cycle. To dissect the functional activity of atALKBH9B in AMV infection, we performed a protein-mapping analysis to identify the putative domains required for regulating this process. In this context, the mutational analysis of the protein revealed that the residues between 427 and 467 positions are critical for in vitro binding to the AMV RNA. The atALKBH9B amino acid sequence showed intrinsically disordered regions (IDRs) located at the N-terminal part delimiting the internal AlkB-like domain and at the C-terminal part. We identified an RNA binding domain containing an RGxxxRGG motif that overlaps with the C-terminal IDR. Moreover, bimolecular fluorescent experiments allowed us to determine that residues located between 387 and 427 are critical for the interaction with the AMV CP, which should be critical for modulating the viral infection process. Finally, we observed that atALKBH9B deletions of either N-terminal 20 residues or the C-terminal’s last 40 amino acids impede their accumulation in siRNA bodies. The involvement of the regions responsible for RNA and viral CP binding and those required for its localization in stress granules in the viral cycle is discussed.

Recent Acquisition of Functional m6A RNA Demethylase Domain in Orchid Ty3/Gypsy Elements

“…This ORF contains a 2-ODD domain, conserves the cofactor-binding residues necessary for its activity (Figure 4B), is most closely related to known RNA demethylases from human and Arabidopsis (Figure 4A) and, most importantly, the purified protein shows RNA demethylase activity in vitro (Figure 5) and capability to bind RNA (Figure 6). Interestingly, the apparent constant dissociation (Kd) calculated for the paALKBH5-like protein (0.21 µM, Figure 6) was similar to that observed for atALKBH9B (0.30 µM) (Alvarado-Marchena et al, 2021) reinforcing the notion that both proteins are functionally equivalent.…”

The m6A RNA Demethylase ALKBH9B Plays a Critical Role for Vascular Movement of Alfalfa Mosaic Virus in Arabidopsis

“…Using the online tool Clustal Omega, we obtained the alignment between the amino acid sequences of the AlkB domain (residues 222 to 412 in ALKBH9B) from these three proteins and human ALKBH5 (hsALKBH5, residues 85 to 284; Figure 1A ). Remarkably, the AlkB domain and some amino acids described as key residues for the m 6 A demethylation activity of hsALKBH5 (highlighted in Figure 1A ) are quite conserved in the three ALKBH9 Arabidopsis co-orthologues ( Feng et al, 2014 ; Lu et al, 2014 ; Alvarado-Marchena et al, 2021 ). Thus, we observed that the known HxD/E·· H and RxxxxxR motifs are conserved between the three proteins ( Figure 1A , highlighted in yellow).…”

“…In previous work, we found that the AMV CP interacts with ALKBH9B and we showed that this protein decreases m 6 A relative abundance along AMV vRNAs. In turn, this promotes viral accumulation in Arabidopsis since the virus showed an impaired systemic invasion capability in alkbh9b plants ( Martínez-Pérez et al, 2017 ; Alvarado-Marchena et al, 2021 ). Here, we discard that the other ALKBH9 close paralogous proteins affect AMV infection and we make a deep analysis of the infection restriction observed in alkbh9b plants.…”

m6A RNA methylation impairs gene expression variability and reproductive thermotolerance inArabidopsis

“…Further to the control at DNA or chromatin level, we suggest that an epitranscriptomic mark adds to the complex regulation of gene expression variability. Given that m 6 A-methylated mRNAs are preferably located to cytoplasmic stress granules (SGs) (Alvarado-Marchena et al, 2021; Fu and Zhuang, 2020; Ries et al, 2019; Scutenaire et al, 2018), we speculate that the physical confinement of m 6 A-methylated transcripts to SGs might play a role in stable gene expression variability. To test this possibility, we compared LVGs and random genes for their SG enrichment score determined in our previous study (Kim et al, 2021), and LVGs indeed showed stronger SG enrichment (Fig.…”