Small nucleolar RNAs (snoRNAs) are critical in guiding post-transcriptional modifications like 2'-O-methylation (Nm) in RNA, which play crucial roles in downstream processes such as splicing and translation. This study provides a novel method for Nm validation, addressing a significant gap in modern Nm research, and oPers insight into the intricacies of snoRNA-guided Nm. While mapping of Nm modifications has seen significant improvement within the past decade, no major techniques have been able to validate these potential sites. Additionally, many mapping techniques lack consensus among proposed Nm sites, especially on less abundant species such as mRNAs. Without a proper validation technique, Nm research lags compared to its peer post-transcriptional modifications. The RNaseH-based Nm-VAQ assay proposed here quantifies 2'-O- methylation at single nucleotide resolution across various RNA species including rRNA, snRNA, and mRNA. Its optimization for mRNA allows for an unprecedented way to study the ePects of Nm modifications in low abundance transcripts. This allows researchers to validate proposed Nm sites and understand the stoichiometry of methylated versus unmethylated sites and its downstream ePects on translation. This study also explores the use of synthetic snoRNAs in guiding Nm modifications. Utilizing the novel Nm-VAQ validation assay, exogenous snoRNAs are shown to rescue Nm in genetic knockout models. These exogenous snoRNAs can be modified to guide Nm at any location along the target RNA transcript. This method facilitates the study of how Nm modifications on diPerent regions of an mRNA transcript, including but not limited to the start/stop codon, 5' or 3' UTR, exons, and splice donor/acceptor sites, impact downstream processes. Preliminary work indicates that synthetically modified snoRNAs demonstrate the ability to modify exogenous RNA transcripts such as luciferase, impacting translation ePiciency and protein expression. The addition of a modification in the luciferase exon increases mRNA abundance but decreases protein expression, consistent with previous findings on other mRNAs. Future work is needed using the Nm-VAQ assay to fully elucidate the relationship between Nm modification levels and the corresponding changes in transcription and translation. These findings set the scene for novel understanding of the relationship between snoRNA abundance, 2'-O-methylation ePiciency, and Nm's impact on gene expression. Enhancement of translation in living cells through synthetic ribonucleoprotein granule

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