snoRNA-guided ribosome heterogeneity in a model of NSCLC collective invasion

The processing, folding, and chemical modification of ribosomal RNAs (rRNAs) are critical for proper ribosome assembly and function and are tightly coordinated by a complex network of proteins and non-coding RNAs (ncRNAs). Among these, an abundant group of ncRNAs, small nucleolar RNAs (snoRNAs), guide the folding and site-specific modification of over 200 nucleotides in human rRNAs. Emerging evidence has linked changes in rRNA modification pattern to distinct tumor stages, but how rRNA modification heterogeneity manifests and contributes to cancer progression remains unknown. One mechanism driving cancer progression to metastasis is collective invasion, which is defined by the presence of cooperative subpopulations within a tumor. Previous work by the Marcus lab isolated two cell subpopulations, leaders and followers, from the H1299 cell line based on their distinct phenotypes. To assess the role of snoRNA-guided modification heterogeneity in invasion, we applied an unbiased genome-wide sequencing approach to leader and follower subpopulations to map rRNA modifications and snoRNAs. We identified 418 snoRNAs in leaders and followers. Of the differentially expressed snoRNAs, 60% are predicted to be ribosome targeting. Analysis of the snoRNA-guided rRNA modifications, revealed differential modification status at specific sites on the rRNA between leaders and followers. The majority of the top differentially modified sites are among the known dynamically modified sites across different cancers and tissues. While the sites of modification heterogeneity are not localized to a distinct site on the ribosome, they fall within key functional regions near the decoding center, subunit bridges, within the L1 stalk, and near the binding site of key ribosomal proteins. Overall, a weakly positive Spearman correlation of <0.3 between snoRNA abundance and rRNA modification status suggests snoRNA abundance alone does not inform the global rRNA modification pattern. To uncouple the roles of snoRNA expression and rRNA modification, we apply a set of biochemical tools and demonstrate that leaders and followers have differential growth and translation capacities that can be changed by altering snoRNA expression. Collectively, our data reveal novel modulators of growth and translation in NSCLC and shed light on the molecular principles underlying ribosomal modification heterogeneity in invasive cancer cells.