Comparative analyses suggest a link between mRNA splicing, stability, and RNA covalent modifications in flowering plants.

BACKGROUND: In recent years, covalent modifications on RNA nucleotides have emerged as pivotal moieties influencing the structure, function, and regulatory processes of RNA Polymerase II transcripts such as mRNAs and lncRNAs. However, our understanding of their biological roles and whether these roles are conserved across eukaryotes remains limited. RESULTS: In this study, we leveraged standard polyadenylation-enriched RNA-sequencing data to identify and characterize RNA modifications that introduce base-pairing errors into cDNA reads. Our investigation incorporated data from three Poaceae (Zea mays, Sorghum bicolor, and Setaria italica), as well as publicly available data from a range of stress and genetic contexts in Sorghum and Arabidopsis thaliana. We uncovered a strong enrichment of RNA covalent modifications (RCMs) deposited on a conserved core set of nuclear mRNAs involved in photosynthesis and translation across these species. However, the cohort of modified transcripts changed based on environmental context and developmental program, a pattern that was also conserved across flowering plants. We determined that RCMs can partly explain accession-level differences in drought tolerance in Sorghum, with stress-associated genes receiving a higher level of RCMs in a drought tolerant accession. To address function, we determined that RCMs are significantly enriched near exon junctions within coding regions, suggesting an association with splicing. Intriguingly, we found that these base-pair disrupting RCMs are associated with stable mRNAs, are highly correlated with protein abundance, and thus likely associated with facilitating translation. CONCLUSIONS: Our data point to a conserved role for RCMs in mRNA stability and translation across the flowering plant lineage.

Transfer learning enables identification of multiple types of RNA modifications using nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

RNA N6-Methyladenosine Affects Copper-Induced Oxidative Stress Response in Arabidopsis thaliana.

Recently, post-transcriptional regulation of mRNA mediated by N6-methyladenosine (m6A) has been found to have profound effects on transcriptome regulation during plant responses to various abiotic stresses. However, whether this RNA modification can affect an oxidative stress response in plants has not been studied. To assess the role of m6A modifications during copper-induced oxidative stress responses, m6A-IP-seq was performed in Arabidopsis seedlings exposed to high levels of copper sulfate. This analysis revealed large-scale shifts in this modification on the transcripts most relevant for oxidative stress. This altered epitranscriptomic mark is known to influence transcript abundance and translation; therefore we scrutinized these possibilities. We found an increased abundance of copper-enriched m6A-containing transcripts. Similarly, we also found increased ribosome occupancy of copper-enriched m6A-containing transcripts, specifically those encoding proteins involved with stress responses relevant to oxidative stressors. Furthermore, the significance of the m6A epitranscriptome on plant oxidative stress tolerance was uncovered by assessing germination and seedling development of the mta (N6-methyladenosine RNA methyltransferase A mutant complemented with ABI3:MTA) mutant exposed to high copper treatment. These analyses suggested hypersensitivity of the mta mutant compared to the wild-type plants in response to copper-induced oxidative stress. Overall, our findings suggest an important role for m6A in the oxidative stress response of Arabidopsis.

PELOTA and HBS1 suppress co-translational messenger RNA decay in Arabidopsis.

Various messenger RNA (mRNA) decay mechanisms play major roles in controlling mRNA quality and quantity in eukaryotic organisms under different conditions. While it is known that the recently discovered co-translational mRNA decay (CTRD), the mechanism that allows mRNAs to be degraded while still being actively translated, is prevalent in yeast, humans, and various angiosperms, the regulation of this decay mechanism is less well studied. Moreover, it is still unclear whether this decay mechanism plays any role in the regulation of specific physiological processes in eukaryotes. Here, by re-analyzing the publicly available polysome profiling or ribosome footprinting and degradome sequencing datasets, we discovered that highly translated mRNAs tend to have lower co-translational decay levels. Based on this finding, we then identified Pelota and Hbs1, the translation-related ribosome rescue factors, as suppressors of co-translational mRNA decay in Arabidopsis. Furthermore, we found that Pelota and Hbs1 null mutants have lower germination rates compared to the wild-type plants, implying that proper regulation of co-translational mRNA decay is essential for normal developmental processes. In total, our study provides further insights into the regulation of CTRD in Arabidopsis and demonstrates that this decay mechanism does play important roles in Arabidopsis physiological processes.