pCLASH: Mapping snoRNA-guided 2’-O-Methylation Targets on pre-mRNA
Principal Investigator: Christopher Holley, MD, PhD
Research Mentor: Brittany Elliott, PhD
GCS Faculty Advisor: Charles Gersbach, PhD
Abstract: Small nucleolar RNAs (snoRNAs) are noncoding RNAs canonically known to guide post-transcriptional modifications on ribosomal RNAs. It has been previously shown that box C/D snoRNAs are capable of guiding 2’-O-methylations (Nm) on mRNAs, with these modifications regulating physiologic gene expression and protein translation. To date, the comprehensive, transcriptome-wide set of mRNA targets of snoRNAs has not been identified. Here, we demonstrate pCLASH, a method to map the global set of pre-mRNA targets of Nm modification by snoRNAs through crosslinking, ligation, and sequencing of hybrids. Particularly novel aspects of this method include the use of phenol-toluol extraction (PTex) to isolate crosslinked RNA-protein along with the combination of nuclei isolation and rRNA depletion prior to immunoprecipitation. Over 2900 hybrids were obtained from pCLASH, with 166 of them being snoRNA-mRNA hybrids. Of note, the snoRNA-mRNA hybrids/Total Reads ratio using pCLASH was several orders of magnitude higher than previously published methods to identify snoRNA-mRNA interactions. Interestingly, numerous mitochondrial targets were revealed, meriting future explorations regarding the role of snoRNA-guided Nm on mitochondrial function and oxidative phosphorylation.
Full Thesis: Choi-GCS Thesis Final
GCS Reflection: Choi-GCS Reflection Paper
