3.1. m6A Methylation of Nuclear-Derived RNAs Related to Chloroplast Functions
Recent data revealed that m
6A methylation marks in nuclear-derived RNAs are often related to chloroplast function, thus we will provide a brief overview of the nuclear/cytoplasmic m
6A methylome with a focus on this organelle (
Figure 2). With respect to RNA methylation, such as the most widespread adenosine methylation at the N6 position (m
6A), the mainly nuclear localized modifiers (methyltransferases and demethylases) and mostly cytoplasmic interpreters are called writers, erasers, and readers, respectively
[36]. The nuclear m
6A writer complex consists of several conserved and essential components in eukaryotes, including the methyltransferases MTA and MTB, the splicing factor FKBP12 Interacting Protein37 (FIP37), VIRILIZER (VIR), and the conserved E3 ubiquitin ligase HAKAI
[37]. DRS and miCLIP were recently applied to examine the epitranscriptome of the leaky
vir-1 mutant defective in nuclear m
6A methylation and VIR-complemented lines in Arabidopsis
[32]. Frequent cleavage and polyadenylation of the mRNA encoding a chloroplast envelope-bound plant homeodomain transcription factor (PTM) with transmembrane domains was found. PTM (AT5G35210) resides in the outer chloroplast membrane and was suggested to be involved in retrograde signaling upon cleavage of a C-terminal transmembrane domain that sequesters it to the chloroplast
[32][38]. Cleavage and polyadenylation of the
ptm intron 10 terminates transcription prior to a sequence encoding the transmembrane domain, consequently bypassing established retrograde control
[32]. 17,491 sites with restored m
6A modifications in the VIR-complemented line were identified. The AAm
6ACU and AAm
6ACA motifs were confirmed to be the most frequently detected m
6A marks in Arabidopsis
[39][40][41]. Several sites associated with the motif AGm
6AUU were also detected, raising the possibility that a C following m
6A is not a constant feature of the Arabidopsis m
6A code
[32]. The preferential 3’ UTR localization of m
6A in cytoplasmic mRNAs was also confirmed. The differential error sites were exclusively found in this region and no enrichment over stop codons was identified using both, miCLIP or DRS. Strikingly, the impact of m
6A loss on pre-mRNA processing was determined and a clear defect in RNA 3’ end formation in
vir-1 was observed
[32]. 3579 genes with an altered 3’ position profile in the
vir-1 mutant were identified. For instance, the
prpl34 mRNA, which encodes a chloroplast ribosomal protein, is methylated in at least two positions in the 3’ UTR and displays an increase in alternative polyadenylation at a proximal poly(A) site in the
vir-1 mutant. These findings suggest that changes in 3’ end poly(A) position of RNAs in the
vir-1 mutant may result directly from the loss of m
6A and implies a crucial role of the cellular m
6A methylome in plastid gene expression.
Figure 2. Cellular scenario of the RNA methylome in plants. m6A methylation and demethylation of nuclear-derived RNAs mainly takes place in the nucleus close to start and stop codons and in the 3’ UTRs and participate in the processing, stability, and localization of RNAs whereas m6A readers function mostly in the cytoplasm. m6A methylated transcripts predominantly encode chloroplast proteins important for gene expression, photosynthesis, and other plastid functions. Only three 16S rRNA writers–PFC1 for m6A, CMAL for m4Cm, and RsmD for m2G methylations-have been described in chloroplasts but writers for mRNAs, as well as erasers and readers are entirely unknown. The activity of these modifiers and interpreters is presumably crucial for the fate of transcripts important for plant development, stress responses, and acclimation processes upon environmental changes. Red stars: RNA methylation marks; orange clouds: readers.
Conflicting results in the Arabidopsis epitranscriptome, such as the enrichment of the m6A distribution within the plant mRNA molecules, can be solved using state-of-the-art approaches and new technologies. Still, it is important to restate that the effects of m6A are challenging and possibly condition-dependent. To date, many chloroplast-related, nucleus encoded transcripts that carry m6A were identified. Regardless of nucleus- or chloroplast-derived transcripts, a compelling connection between m6A RNA modifications and chloroplast and/or acclimation functions in plants seems to be clear. Despite the obvious progress summarized here, the exact understanding of how m6A modifications regulate the function of nucleus-encoded RNAs that encode chloroplast proteins remains a future challenge (Figure 2).
In general, the m
6A modification is subjected to dynamic regulation in both development and response to cellular stimuli and ever-changing conditions in eukaryotes
[25][42]. Although m
6A appears to be the most abundant internal RNA modification of plants, the m
6A pattern and its regulation in humans is by far much better investigated than in plants
[37]. Importantly, a relatively high proportion of nuclear/cytoplasmic transcripts encoding photosynthesis-related proteins have been shown to undergo m
6A modifications implying important roles in chloroplast functions. The m
6A modifications generally play critical roles in many areas of the plant life
[42][43]. Similar to the animal system the m
6A:A ratio is 1.5% in young Arabidopsis seedlings
[44]. Elucidation of the function of the m
6A RNA modification is a challenging and growing field in plant RNA research
[45]. Rapidly evolving methodological approaches will allow us to increase our understanding about the function and regulation of m
6A in plants, which certainly will contribute to improve our knowledge about cellular functions, developmental cycles, and acclimation processes related to chloroplast functions.
The m
6A methylome in plants was first identified in two accessions of
A. thaliana, Can-0 and Hen-16, two wild-collected lines from areas that vary drastically in photosynthetically active radiation values. However, the m
6A modifications were shown to be remarkably conserved across these two lines. Surprisingly, m
6A was enriched not only within the 3’ UTRs and stop codons but also around the start codons, a feature only observed in plant RNAs
[39]. The consensus recognition sequence of nuclear/cytoplasmic transcripts has been described as RRm
6ACH in the epitranscriptome of mammals, where R = G > A and H = U > A > C
[26][46]. Interestingly, in plant RNAs new conserved motifs were found, indicating the presence of distinct target sequence motifs for m
6A target-methylations
[21][39]. For example, the URUAY (R = G > A,Y = U > A) m
6A methylation motif is plant specific and was shown to have a role in RNA stabilization
[47]. In both, Can-0 and Hen-16, gene ontology unveiled many biological pathways related to chloroplast functions. In particular, more than 60% of cytoplasmic transcripts containing m
6A in both, start and stop codons and about 40% of those carrying the modification only in the start site were highly associated with photosynthetic functions. A complete list can be accessed in a previous work
[39].
Differential m
6A patterns of cytoplasmic transcripts across different organs were also investigated in Arabidopsis
[40]. More than 70% of the transcripts were m
6A modified in leaves, flowers, and roots. The consensus sequence RRm
6ACH was found in over 75% of the transcripts, but only one dominant peak of m
6A enrichment was identified around the 3’ UTR and stop codons in the Arabidopsis transcriptome. Notably, all three organs analyzed share about 290 m
6A-methylated transcripts and their coding proteins are mostly located in the chloroplast. Most interestingly, differential m
6A methylation among leaves, flowers, and roots showed that green leaves had the highest extent of m
6A methylation among the three organs. These transcripts are mainly related to photosynthesis, regulation of transcription, and stress response. Highly methylated transcripts presented in leaves and roots had specific functions related to the respective organs—photosynthesis in leaves and transport in roots
[40].
The extent of m
6A methylation was also compared to the levels of the respective transcripts in three organs of Arabidopsis. Most of the highly expressed transcripts were less modified by m
6A when compared with transcripts expressed at low level. This observation implies an important function of m
6A in regulation of RNA levels and/or stability in plant cells. Low level transcripts may require a relatively higher extent of m
6A modifications to maintain RNA stability in the cells and vice versa
[40]. A role of m
6A in the stabilization of mRNAs under salt stress has been reported in Arabidopsis. In this case, m
6A is dynamically added to salt-stress-related transcripts to protect RNAs from degradation
[48]. The process of flowering, for instance, is delayed in
alkbh10b mutant plants, which lack an m
6A eraser. This phenotype can be explained by the destabilization of transcripts involved in flowering transition when m
6A modifications are not reversed
[49]. Therefore, the exact mechanisms of regulation involving m
6A in plants are far from being identified. Why under certain conditions m
6A stabilizes or destabilizes a specific transcript is still to be determined. So far, the dependence of the stability of nuclear-encoded RNAs associated with plastid functions also remains an open question. Whether and how m
6A methylation in the nucleus/cytoplasmic system reshapes the proteome or modulates gene expression within the chloroplast and vice versa remains elusive.
3.2. General Aspects on m6A Methylation Marks in Chloroplast RNAs
Virtually nothing is known about m
6A epitranscriptome players in chloroplasts. In contrast to nuclear/cytoplasmic systems, only one chloroplast m
6A RNA writer has been described
[50] but erasers and readers are yet to be discovered (
Figure 2). The m
6A methylome was studied in Arabidopsis chloroplasts and mitochondria
[42][51]. mtRNAs in both Arabidopsis and cauliflower undergo N6-adenosine methylation modifications with an occurrence of about 4–5 m
6A sites per 1000 adenosine residues. Several m
6A modifications were detrimental for translation, while a single modification in the start codon suggested an enhancement in the translatability of the mitochondrial transcript
[51].
Remarkably, chloroplast transcripts are highly m
6A methylated, implying important roles in photosynthesis and/or plastid gene expression
[42][43]. Over 98% of chloroplast transcripts were chemically modified by m
6A, which is by far much more than the modification status found in the nuclear transcriptome (73%). Furthermore, about 4.6 to 5.8 m
6A sites per transcript were found in the chloroplast but only about 1.4 to 2.0 sites per transcript in the cytoplasm, again emphasizing an important link between m
6A modifications and chloroplast functions
[41]. The most modified transcripts found in this analysis were associated with chloroplast rRNA and mRNAs.
The previously observed dominant m
6A enrichment within the 3´ UTR and near stop codons in nuclear-derived mRNAs was not observed in the chloroplast. Instead, m
6A peaks were found evenly distributed in chloroplast transcripts with higher methylations in exons when compared to introns, suggesting that the regulatory mechanisms may be different between the nucleus/cytoplasm and chloroplast systems. The translation and stability of chloroplast transcripts are commonly regulated by factors acting on 5’ and 3’ UTRs. In addition, degradation of plastid RNAs is thought to be initiated by endonucleolytic cleavages
[4][52]. Thus, it is likely that m
6A methylation in conjunction with RNA-binding proteins controls the fate of mRNAs in the plant organelle at multiple levels.
Most surprisingly, many m
6A consensus sequences in the chloroplasts and mitochondria share homology to those in mammals and plant nuclear transcritptomes, indicating an evolutionary related process
[41][51]. The two most common sequence motifs found in the Arabidopsis chloroplast transcriptome were GGm
6ACC and GGm
6ACU. The methylation extent compared to RNA levels in the chloroplast was similar to that found in the nuclear-derived RNAs in Arabidopsis, as most of the highly expressed plastid transcripts were less modified by m
6A, and vice versa, corroborating a related or analogous development. However, tissue-specific deviations were also observed. For example, in root amyloplasts the moderately expressed transcripts were more methylated and those expressed at lower or higher levels carried less m
6A modifications
[41].