N⁶-methyladenosine (m⁶A) emerges as an important modification in eukaryotic mRNAs. m⁶A has first been reported in 1974, and its functional significance in mammalian gene regulation and importance for proper development have been well established. An arsenal of writer, eraser, and reader proteins accomplish deposition, removal, and interpretation of the m⁶A mark, resulting in dynamic function. This led to the concept of an epitranscriptome, the compendium of RNA species with chemical modification of the nucleobases in the cell, in analogy to the epigenome. While m⁶A has long been known to also exist in plant mRNAs, proteins involved in m⁶A metabolism have only recently been detected by mutant analysis, homology search, and mRNA interactome capture in the reference plant Arabidopsis thaliana. Dysregulation of the m⁶A modification causes severe developmental abnormalities of leaves and roots and altered timing of reproductive development. Furthermore, m⁶A modification affects viral infection. Here, we discuss recent progress in identifying m⁶A sites transcriptome-wide, in identifying the molecular players involved in writing, removing, and reading the mark, and in assigning functions to this RNA modification in A. thaliana. We highlight similarities and differences to m⁶A modification in mammals and provide an outlook on important questions that remain to be addressed.

Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress.

Summary: Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine‐tune responses to environmental and developmental cues. Arabidopsis thaliana At‐RS31, a plant‐specific splicing factor in the Serine/Arginine‐rich (SR) protein family, responds to light and the Target of Rapamycin (TOR) signalling pathway, yet its downstream targets and regulatory impact remain unknown. To identify At‐RS31 targets, we applied individual‐nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At‐RS31 mutant and overexpressing plants further revealed its effects on alternative splicing. iCLIP identified 4034 At‐RS31 binding sites across 1421 genes, enriched in CU‐rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the arginine/serine (RS) domain of At‐RS31 may influence its binding specificity in planta , underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At‐RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator. By regulating stress response genes and genes in both TOR and abscisic acid signalling pathways, At‐RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing.

N6-methyladenosine (m ⁶ A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology-domain family (YTHDF) proteins. Here, we show that in Arabidopsis thaliana , the interaction between m ⁶ A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins and their mRNA target sets overlap, with ALBA4 binding sites being juxtaposed to m ⁶ A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m ⁶ A binding to ECT2. Accordingly, both the biological functions of ECT2, and its binding to m ⁶ A targets in vivo, require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m ⁶ A-reader in Arabidopsis , and define a molecular foundation for the concept of facilitated m ⁶ A reading, which increases the potential for combinatorial control of biological m ⁶ A effects.