Abstract Eukaryotic messenger RNAs are extensively decorated with modified nucleotides and the resulting epitranscriptome plays important regulatory roles in cells 1 . Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated 2–5 . However, it is unclear when in their life cycle RNAs become pseudouridylated and what the endogenous functions of mRNA pseudouridylation are. To determine if pseudouridine is added co-transcriptionally, we conducted pseudouridine profiling 2 on chromatin-associated RNA to reveal thousands of intronic pseudouridines in nascent pre-mRNA at locations that are significantly associated with alternatively spliced exons, enriched near splice sites, and overlap hundreds of binding sites for regulatory RNA binding proteins. Multiple distinct pseudouridine synthases with tissue-specific expression pseudouridylate pre-mRNA sites, and genetic manipulation of the predominant pre-mRNA modifying pseudouridine synthases PUS1, PUS7 and RPUSD4 induced widespread changes in alternative splicing in cells, supporting a role for pre-mRNA pseudouridylation in alternative splicing regulation. Consistently, we find that individual pseudouridines identified in cells are sufficient to directly affect splicing in vitro . Together with previously observed effects of artificial pseudouridylation on RNA-RNA 6–8 and RNA-protein 9–11 interactions that are relevant for splicing, our results demonstrate widespread co-transcriptional pre-mRNA pseudouridylation and establish the enormous potential for this RNA modification to control human gene expression.

Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect splicing