MRP
RNA is an abundant, essential
noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP
RNA cause pleiotropic human diseases, including
cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP
RNA locus, thereby attaining what has eluded RNAi and
RNase H experiments: elimination of MRP
RNA in the majority of cells. The resulting accumulation of
ribosomal RNA (
rRNA) precursor-analyzed by
RNA fluorescent in situ hybridization (FISH), Northern blots, and
RNA sequencing-implicates MRP
RNA in
pre-rRNA processing. Amelioration of
pre-rRNA imbalance is achieved through rescue of MRP
RNA levels by ectopic expression. Furthermore, affinity-purified MRP
ribonucleoprotein (RNP) from HeLa cells cleaves the human
pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP
RNA expression restores cleavage activity. Thus, a role for
RNase MRP in human
pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and
RNase H-based degradation.