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Genes Dev. 2017 Jan 1;31(1):59-71. doi: 10.1101/gad.286963.116. Epub 2017 Jan 23.

Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

Author information

1
Department of Chemistry and Biochemistry, BioFrontiers Institute, University of Colorado at Boulder, Boulder, Colorado 80302, USA.
2
Howard Hughes Medical Institute, University of Colorado at Boulder, Boulder, Colorado 80302, USA.

Abstract

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.

KEYWORDS:

CRISPR–Cas9; RMRP; RNase MRP; RNase P; preribosomal RNA processing

PMID:
28115465
PMCID:
PMC5287113
DOI:
10.1101/gad.286963.116
[Indexed for MEDLINE]
Free PMC Article

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