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

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• MRP RNA is a crucial noncoding RNA with poorly understood functions in humans.
• Mutations in the MRP RNA gene are linked to human diseases like cartilage hair hypoplasia (CHH).

Purpose of the Study:

• To elucidate the function of MRP RNA in human cells.
• To establish a role for RNase MRP in pre-ribosomal RNA (pre-rRNA) processing.
• To demonstrate the utility of CRISPR-Cas9 for studying essential noncoding RNAs.

Main Methods:

• CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus.
• RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing to analyze RNA levels.
• In vitro cleavage assays using affinity-purified MRP ribonucleoprotein (RNP).

Main Results:

• CRISPR disruption successfully eliminated MRP RNA in most cells, a feat not achieved by RNAi or RNase H.
• Accumulation of pre-rRNA was observed upon MRP RNA depletion, implicating MRP RNA in pre-rRNA processing.
• MRP ribonucleoprotein (RNP) demonstrated in vitro pre-rRNA cleavage activity, which was lost upon gene editing and restored by ectopic expression.

Conclusions:

• A critical role for RNase MRP in human pre-rRNA processing is definitively established.
• Targeted CRISPR disruption is a powerful method for functional analysis of essential noncoding RNAs resistant to other methods.
• Understanding MRP RNA function may provide insights into cartilage hair hypoplasia and related disorders.