Intron Editing Reveals SNORD-Dependent Maturation of the Small Nucleolar RNA Host Gene GAS5 in Human Cells

The GAS5 gene encodes a long non-coding RNA (lncRNA) and intron-located small nucleolar RNAs (snoRNAs). Its structure, splice variants, and diverse functions in mammalian cells have been thoroughly investigated. However, there are still no data on a successful knockout of GAS5 in human cells, with most of the loss-of-function experiments utilizing standard techniques to produce knockdowns. By using CRISPR/Cas9 to introduce double-strand breaks in the terminal intronic box C/D snoRNA genes (SNORDs), we created monoclonal cell lines carrying continuous deletions in one of the GAS5 alleles. The levels of GAS5-encoded box C/D snoRNAs and lncRNA GAS5 were assessed, and the formation of the novel splice variants was analyzed. To comprehensively evaluate the influence of specific SNORD mutations, human cell lines with individual mutations in SNORD74 and SNORD81 were obtained. Specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Further analysis revealed that SNORD74 contains a specific regulatory element modulating the maturation of the GAS5 precursor transcript. The results demonstrate that the maturation of GAS5 occurs through the m6A-associated pathway in a SNORD-dependent manner, which is a quite intriguing epitranscriptomic mechanism.