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J Biol Chem. 2019 May 3;294(18):7445-7459. doi: 10.1074/jbc.RA118.006271. Epub 2019 Mar 21.

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

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From the Department of Human Genetics, McGill University, Montréal, Québec H3A 0C7, Canada.
the Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec H3T 1E2, Canada.
the Montréal Neurological Institute, McGill University, Montréal, Québec H3A 2B4, Canada.
the Translational Proteomics Laboratory, Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec H2W 1R7, Canada.
Pediatrics, McGill University, Montréal, Québec H3A 0G4, Canada.
the Department of Internal Medicine, Division of Medical Genetics, Montréal Children's Hospital, McGill University Health Center, Montréal, Québec H4A 3J1, Canada.
the Child Health and Human Development Program, and.
MyeliNeuroGene Laboratory, Research Institute, McGill University Health Center, Montréal, Québec H4A 3J1, Canada.
the Departments of Neurology and Neurosurgery and.
the Division of Neurology, Children's Hospital of Philadelphia (CHOP), Philadelphia, Pennsylvania 19104.
the Institute of Metabolic Disease, Baylor Research Institute, Dallas, Texas 75204.
INSERM U1212-CNRS UMR5320, Université de Bordeaux, Bordeaux, France, and.
the Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3T 1J4, Canada.
From the Department of Human Genetics, McGill University, Montréal, Québec H3A 0C7, Canada,


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.


CRISPR/Cas; RNA polymerase III; RNA-seq; brain cytoplasmic 200 RNA (BCYRN1); leukodystrophy; myelin; oligodendrocyte; proteomics; transcription; transfer RNA (tRNA)

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