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

The 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 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 (s), we created monoclonal cell lines carrying continuous deletions in one of the alleles. The levels of -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 mutations, human cell lines with individual mutations in and were obtained. Specific mutations in led to the downregulation of all -encoded SNORDs and GAS5 lncRNA. Further analysis revealed that contains a specific regulatory element modulating the maturation of the precursor transcript. The results demonstrate that the maturation of GAS5 occurs through the m6A-associated pathway in a -dependent manner, which is a quite intriguing epitranscriptomic mechanism.