SnoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification

Small nucleolar RNAs (snoRNA) are non-coding RNAs known for guiding RNA modifications including 2'-O-methylation (Nm) and pseudouridine (?). While snoRNAs may also interact with other RNAs such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome-wide. Here we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues. Many interactions occur outside of snoRNA-guided rRNA modification sites, hinting at non-canonical functions beyond RNA modification. We find that one of these snoRNAs, SNORA73, targets mRNAs that encode secretory proteins and membrane proteins. SNORA73 also interacts with 7SL RNA, part of the signal recognition particle (SRP) required for protein secretion. The mRNA-SNORA73-7SL RNA interactions enhance the association of the SNORA73-target mRNAs with SRP, thereby facilitating secretion of the encoded proteins. Overall design: Sample 1-34 are duplicates for snoKARR-seq experiments detecting RNA-RNA interactions. Homogenized tissue or live cells are labeled with N3-kethoxal, which reacts with cellular RNA close to saturation, followed by formaldehyde-assisted fixing. Physically proximal RNAs labeled by N3-kethoxal are crosslinked by a chemically modified dendrimer bearing an activated alkyne group through the click chemistry. Subsequently, proteins bound to RNAs are digested by proteinase K. Cross-linked RNAs are fragmented and enriched using streptavidin beads. Enriched RNAs undergo proximity ligation to generate chimeric RNAs, after which the kethoxal labeling is reversed. The resulting RNAs are subjected to RT to generate cDNAs. 10% of the cDNA was saved as input samples. Next, a pool of biotinylated ASOs targeting a group of specific snoRNAs is used to enrich cDNAs derived from snoRNAs of interest. Both the input sample and the enriched cDNAs are amplified for high-throughput sequencing using the SMARTer Stranded Total RNA-seq kit v2 pico input mammalian. Sample 35-40 are duplicates for small RNA-seq experiments from cellular small RNA fractions Dot-Bracket Notation is described here: https://wiki.eternagame.org/wiki/Dot-Bracket_Notation