Poster Presentation Australasian RNA Biology and Biotechnology Association 2024 Conference

Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover (#131)

Marco Guarnacci 1 , Pei-Hong Zhang 2 , Madhu Kanchi 1 , Yu-Ting Hung 1 , Hanrong Lin 1 , Nikolay Shirokikh 1 , Li Yang 2 , Thomas Preiss 1 3
  1. EMBL–Australia Collaborating Group, Department of Genome Sciences, John Curtin School of Medical Research, Australian National University, Canberra 2601, Australian Capital Territory, Australia, Canberra, ACT, Australia
  2. CAS Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Scienc, Shanghai, China
  3. Victor Chang Cardiac Research Institute, Sydney, New South Wales, 2010, Australia, Sydney, New South Wales, Australia

Maps of the RNA modification 5-methylcytosine (m5C) often diverge markedly not only due to differences in detection methods, data depth and analysis pipelines, but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m5C site calling pipeline. With the resulting union list of 6,393 m5C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m5C methyltransferases NSUN2 and NSUN6 as the main mRNA m5C ‘writers’, but further showed that the rRNA:m5C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m5C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m5C writers and readers and their effect on mRNA function.