Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200

Karine Choquet,Diane Forget,Elisabeth Meloche,Marie-Josée Dicaire,Geneviève Bernard,Adeline Vanderver,Raphael Schiffmann,Marc R Fabian,Martin Teichmann,Benoit Coulombe,Bernard Brais,Claudia L Kleinman

doi:10.1074/jbc.ra118.006271

Abstract

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3–HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3–HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.

Highlights

We obtained one homozygous mutant clone and two compound heterozygous clones that carry the M852V mutation on one allele and an indel causing a frameshift and premature stop codon on the other allele (Fig. 1a and Fig. S2a). These clones mimic the genotypes observed in POLR3–hypomyelinating leukodystrophy (HLD) cases carrying the c.2554A3 G (p.M852V) mutation, because four out of the five reported patients that are compound heterozygous for this mutation have a premature stop codon on the other allele [3, 5]
We combined CRISPR-Cas9 gene editing and transcriptomic profiling in human cells to uncover polymerase III (Pol III) transcripts that are most vulnerable to POLR3–HLD disease-causing mutations
We found that the POLR3A M852V mutation leads to significantly decreased expression of some but not all Pol III transcripts

Summary

Introduction

Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3– HLD), characterized by deficient central nervous system myelination. We used CRISPR-Cas to introduce the POLR3A mutation c.2554A3 G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. RNA-seq and ChIP-seq datasets generated from HEK293 and MO3.13 cell lines were deposited in the GEO repository under accession number GSE118739. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3– HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease

Methods

Results

Conclusion