RNA can be extensively modified post-transcriptionally with >170 covalent modifications,
expanding its functional and structural repertoire. Pseudouridine (psi), the most
abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA
molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided,
bearing important implications for understanding the physiological target spectrum
of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here,
using a massively parallel reporter based strategy we simultaneously interrogate psi
levels across hundreds of synthetic constructs with predesigned complementarity against
endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation
can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies,
and is constrained by mRNA localization, snoRNA expression levels and the length of
the snoRNA:mRNA complementarity stretches. We exploited these insights for the design
of snoRNAs targeting pseudouridylation at premature termination codons, which was
previously shown to suppress translational termination. However, in this and follow-up
experiments in human cells we observe no evidence for significant levels of readthrough
of pseudouridylated stop codons. Our study enhances our understanding of the scope,
'design rules', constraints and consequences of snoRNA-mediated pseudouridylation.
