Comparative Analyses of RNA Modifications using Nanopore direct long-read RNA in Diverse Brassicaceae Species

Recent advances in direct RNA (dRNA) sequencing have enabled transcriptome-wide detection of RNA modifications, offering new insights into the study of the epitranscriptome. Using Arabidopsis thaliana and four diverse Brassicaceae species, we investigated the biological roles of N6-methyladenosine (m6A) and pseudouridine (pseU). Modification sites were identified using a neural network algorithm and tools such as Modkit, enabling downstream analysis of sequence motifs, exon structure, transcript stability, and relationships between poly-adenylated tail length, modification density, and expression. Highly m6A-modified transcripts were found to be more abundant within crucial cellular functions, while transcripts with shorter poly-A tails showed greater variability in decay rate and expression. These findings challenge the assumptions that longer tails correlate to greater stability, prompting the need to re-evaluate past studies where transcriptome-wide analyses were unavailable. Collectively, these results help to reveal complex regulatory roles for RNA modifications in plants and underscore the benefit of dRNA-seq for comparative epitranscriptomic studies.

I am incredibly grateful to have spent the summer conducting research in the Nelson Stress RNA Lab at the Boyce Thompson Institute, supported by an NSF-funded Research Experience for Undergraduates (REU). Coming from a background primarily in human-focused genomic research, I was initially unsure how transferable that experience would be. I quickly discovered that the bioinformatics skills I had developed were not only beneficial but essential for uncovering questions in plant transcriptomics. Throughout this program, I significantly expanded my experience in bioinformatics and RNA biology. Under the mentorship of Dr. Kyle Palos, I developed computational pipelines to identify RNA covalent modifications (RCMs) across four species in the Brassicaceae family. My project focused on uncovering mechanisms of post-transcriptional regulation involving m6A methylation and pseudouridylation, with the ultimate goal of understanding how these modifications contribute to gene expression and stress response from exposure to environmental conditions. This experience has been pivotal in shaping my research trajectory. It not only provided a strong foundation for a future in transcriptomics, but also gave me the confidence to collaborate across disciplines and critically evaluate scientific literature. I now see computational biology not just as a skill, but as an essential tool to investigate the hidden layers of epitranscriptomic regulation.