Characterization of transcriptomic and epitranscriptomic states between nuclear and cytoplasmic RNA using Nanopore-seq

Epitranscriptomics is a rapidly growing field that studies the chemical modifications of RNA molecules that can alter RNA structure, function, and interaction with other molecules within cells. The recent development of Nanopore-seq has enabled the sequencing of native RNA long-reads, which offers a powerful tool for investigating the dynamic deposition of RNA modifications. An intriguing topic of research relates to the epitranscriptional differences between the nuclear and cytoplasmic cellular compartments.

The primary research objective of our lab is to characterize the transcriptomic and epitranscriptomic alterations between nuclear and cytoplasmic transcripts through the utilization of nanopore sequencing technology.

To address the primary research objective, we use an optimized nuclei isolation protocol of which we isolate nuclear and cytoplasmic RNA. For transcriptomic analyses we employ deep cDNA sequencing to characterize transcripts between nucleus and cytoplasm, including identification of differentially enriched transcripts (DETs), their splicing variants, and polyadenylation tail length. For epitranscriptomic study, we employ direct native RNA Nanopore-seq to detect novel RNA modification features between nuclear and cytoplasmic transcripts. In this aspect, we will focus on rRNA biogenesis pathway since it initiates in the nucleolus (within the nucleus) where the pre-rRNA is transcribed and undergoes a series of processing steps including cleavage, modification, and assembly to generate mature rRNA.


Development of “real-time” transcriptomic and epitranscriptomic analysis tools

Real-time analysis tools have great potential for academic and clinical applications, as they allow for data acquisition and analysis to occur simultaneously during sequencing. Our group is interested in developing tools for real-time analysis using nanopore sequencing. one of our latest developments is the development of NanopoREaTA, a tool that enables the detection of transcriptional changes between distinct conditions in real-time during sequencing (Wierczeiko et al. 2022). We are constantly exploring new avenues to enhance the capabilities of Nanoporeata and other tools to enable even more precise and powerful real-time analysis.

See more:

Anna Wierczeiko, Stefan Pastore, Stefan Mündnich, Mark Helm, *Tamer Butto, *Susanne Gerber. “NanopoReaTA: a user-friendly tool for nanopore-seq real-time transcriptional analysis”. Bioinformatics (2022)