Decoding the epitranscriptome at single molecule resolution
The dynamic deposition of chemical modifications into RNA is a crucial regulator of temporal and spatial 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, as it is in principle capable of mapping all RNA modifications simultaneously, in a quantitative manner, and in full-length native RNA reads. Thus, this technology can drastically boost our understanding of RNA modifications, collectively known as the 'epitranscriptome', at an unprecedented resolution.
In our laboratory, we have recently shown that N6-methyladenosine (m6A) can be identified using direct RNA nanopore sequencing, both in vitro and in vivo [1]. Here, I will present our latest results on how epitranscriptome 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 [2]. We now aim to apply these methods to better understand the biological functions and dynamics of the epitranscriptome, as well as how and why epitranscriptomic dysregulation is often associated to human disease [3].
References:
[1] Liu H*, Begik O*, Lucas MC, Ramirez JM, Mason CE, Wiener D, Schwartz S, Mattick JS, Smith MA and Novoa EM. Accurate detection of m6A RNA modifications in native RNA sequences. Nature Comm 2019, 10:4079. doi:10.1038/s41467-019-11713-9
[2] Begik O*, Lucas MC*, Ramirez JM, Milenkovic I, Cruciani C, Vieira HGS, Medina R, Liu H, Sas-Chen A, Mattick JS, Schwartz S and Novoa EM. Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing. bioRxiv 2021, 189969.
[3] Begik O, Lucas MC, Ramirez JM, Liu H, Mattick JS and Novoa EM. Integrative analyses of the RNA modification machinery reveal tissue- and cancer-specific signatures. Genome Biology 2020, 21:97. doi: 10.1186/s13059-020-02009-z