The regulation of the transcriptome is key to cellular processes that underpin cell biology, development and tissue function. All classes of cellular RNA are subject to post-transcriptional modification, be it by direct chemical modification, editing or non-templated nucleotide additions. Now in its fourth edition, the ‘Epitranscriptome’ meeting first held at EMBL in 2016 (and repeated in 2018 then in 2022 as a virtual conference) was the first RNA modification-dedicated meeting with the focus on mammalian systems. It is now established that biochemical modifications of nucleotides regulate RNA metabolism, modulate cell functions and contribute to severe human diseases including cancer and metabolic disorders. Moreover, the power of single, chemically modified nucleotides to alter the electrostatic charge, base pairing and stability of RNA molecules is exploited for clinical use of stable artificial RNA transcripts such as mRNA vaccines or synthetic small RNA molecules to increase or decrease the expression of therapeutic proteins.