Post-transcriptional modification of
RNA nucleosides occurs in all living organisms.
Pseudouridine, the most abundant modified
nucleoside in non-coding RNAs, enhances the function of
transfer RNA and
ribosomal RNA by stabilizing the
RNA structure. Messenger RNAs were not known to contain
pseudouridine, but artificial pseudouridylation dramatically affects
mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring
mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-
nucleotide-resolution method for
pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved
pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in
mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum
starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible
mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of
pseudouridine synthases implicated in human disease.