Abstract
We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.
Highlights
There is much still to be discovered about the post-transcriptional control of gene expression [1,2]
We developed pseudouridine site identification sequencing (PSI-seq), a high-throughput sequencing technique based on the cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) reverse-transcription stop assay to identify pseudouridines in mRNAs, which we used to systematically characterize the pseudouridine content of the yeast transcriptome
Because the two strongest candidate pseudouridine sites derived from two genes with clear functional roles in translation, we performed gene ontology (GO) analysis to search for functional themes that were enriched among the genes we identified as harboring pseudouridylation sites
Summary
There is much still to be discovered about the post-transcriptional control of gene expression [1,2]. One relatively unexplored mechanism for post-transcriptional regulation of gene expression is chemical modification of mRNAs [3,4]. While nucleotide modifications in non-coding RNAs are well known, relatively little is known about possible modifications in mRNAs, and even less is known about what roles these modifications could play. Methylation of the N6 position of specific adenosines in mRNA is essential for sporulation in S. cerevisiae [11,12]. The role this modification plays in gene expression is still a subject of debate. Multiple studies have shown N6-methyladenosine has an effect on binding of RNA binding proteins [9,13,14], but there are conflicting results on the effect of N6-methyladenosine on mRNA translation [15,16] and on stability of modified transcripts [4,14,17]
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