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

Pseudouridine is a modified nucleotide that is prevalent in human mRNAs and is dynamically regulated. Here, we investigate when in their life cycle mRNAs become pseudouridylated to illuminate the potential regulatory functions of endogenous mRNA pseudouridylation. Using single-nucleotide resolution pseudouridine profiling on chromatin-associated RNA from human cells, we identified pseudouridines in nascent pre-mRNA at locations associated with alternatively spliced regions, enriched near splice sites, and overlapping hundreds of binding sites for RNA-binding proteins. In vitro splicing assays establish a direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. We validate hundreds of pre-mRNA sites as direct targets of distinct pseudouridine synthases and show that PUS1, PUS7, and RPUSD4-three pre-mRNA-modifying pseudouridine synthases with tissue-specific expression-control widespread changes in alternative pre-mRNA splicing and 3' end processing. Our results establish avast potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.

Automated summary

This study investigates the pseudouridylation of mRNAs in their life cycle and reveals its potential regulatory functions. By analyzing chromatin-associated RNA from human cells, pseudouridines were found in nascent pre-mRNA at regions associated with alternative splicing and near splice sites. In vitro splicing assays demonstrate the direct effect of individual endogenous pre-mRNA pseudouridines on splicing efficiency. The study establishes a potential for cotranscriptional pre-mRNA pseudouridylation to regulate human gene expression via alternative pre-mRNA processing.