Decoding the epitranscriptome at single-molecule resolution

Eva Maria Novoa

Eva Maria Novoa, Centre for Genomic Regulation, Spain


The dynamic deposition of chemical modifications into RNA is a crucial regulator of temporally and spatially accurate gene expression programs. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each RNA modification individually. In this context, direct RNA nanopore sequencing (dRNAseq) has emerged as a promising technology that can overcome these limitations. In our laboratory, we have shown that N6-methyladenosine (m6A) can be identified in the form of systematic basecalling 'errors' using direct RNA nanopore sequencing, both in vitro and in vivo. Here, I will present our latest results on how diverse epitranscriptomic marks (Y, Am, Um, Gm, m5C, etc.) as well as their dynamics can be studied in rRNAs, snRNAs, and mRNAs, in individual RNA molecules. Contrary to expectations, we find that none of the environmental stresses tested lead to significant changes in yeast ribosomal RNAs. By contrast, our method does recapitulate previously reported heat-dependent pseudouridine (Y) modifications in snRNA and snoRNAs. Moreover, we report novel heat-sensitive Y mRNA modifications, as well as identify previously unreported Y modifications in mitochondrial rRNAs. Finally, I will discuss our latest results to estimate per-site modification stoichiometries from individual RNA molecules, allowing us to quantify the RNA modification stoichiometry changes between two conditions. Altogether, our results show that RNA modification dynamics can be studied in individual RNA molecules, providing novel avenues and tools to better study the biological functions and dynamics of the epitranscriptome, as well as to decipher how and why epitranscriptomic dysregulation is often associated to human disease.


Eva Maria Novoa is group leader at the Center for Genomic Regulation (CRG) in Barcelona Spain, where she leads the Epitranscriptomics and RNA Dynamics laboratory. Her lab is focused on deciphering the language of RNA modifications, collectively known as the epitranscriptome, and how its orchestration can regulate our cells in a space-, time-, and signal-dependent manner.