Poster Presentation 50 Years Shine-Dalgarno Symposium 2023

Profiling messenger RNA modifications associated with translation in B-cell acute lymphoblastic leukemia. (#144)

Favour Oluwapelumi Oyelami 1 2 3 , Amna Chowdhery 2 , Agin Ravindran 1 2 3 , Aditya Sethi 1 2 3 , Katrina Woodward 2 , Pablo Acera Mateos 1 2 3 , Shafi Mahmud 2 , Madhu Kanchi 2 , Nikolay Shirokikh 2 , Eduardo Eyras 1 2 3
  1. EMBL Australia Partner Laboratory Network at the Australian National University, Canberra, Australian Capital Territory (ACT), Australia
  2. The Shine-Dalgarno Centre for RNA Innovation, The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory (ACT), Australia
  3. The Centre for Computational Biomedical Sciences, The John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory (ACT), Australia

RNA modifications,(epitranscriptome), have been shown to play crucial roles in mRNA translation, stability, and degradation, are emerging as important therapeutic targets, and are essential in designing messenger (mRNA) therapeutics and vaccines. However, the specific code of modifications that enhance or repress translation remains unknown. Unveiling this code will be critical to understanding disease processes and improving the design of therapeutic mRNA. Here, we investigate the mRNA modifications in B-cell acute lymphoblastic leukemia (B-ALL) in association with translation.  

We applied our tool, CHEUI (CH3 (methylation) Estimation Using Ionic current), to simultaneously detect N6-methyladenosine (m6A) and 5-methylcytidine (m5C) modifications in mRNAs from two B-ALL cell models using nanopore direct RNA sequencing (DRS). Using high-confidence m6A and m5C sites in mRNA detected by CHEUI, we recover the profiles identified previously using other techniques in different cell models. Our analysis revealed that m6A modifications were enriched in the 3'UTR region, consistent with previous findings, while m5C modifications were more prevalent in the 5’UTR region. We also identified enriched DRACH/RRACH motifs in the detected m6A sites, confirming our identified sites in B-ALL cells. 

To investigate the potential role of m6A and m5C modifications in mRNA translation, we compared their profiles between whole-cell and polysomal fractions. We identified modified sites that presented either increased or decreased modification stoichiometry in the polysomal fraction, indicating potential mechanisms of translation regulation. Moreover, using gene set enrichment analysis, we identified gene pathways associated with differential RNA methylation and active translation in B-ALL cells.  

Our results demonstrate the potential of using CHEUI and DRS to provide accurate and comprehensive detection of RNA modifications, highlights the biomedical importance of profiling RNA modifications in disease contexts to gain insight into the role of epitranscriptomics in disease pathology and provide valuable information needed in the development of epitranscriptome-based RNA drugs and mRNA therapeutics.