The leukodystrophy mutation Polr3b R103H causes homozygote mouse embryonic lethality and impairs RNA polymerase III biogenesis

Abstract

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3a G672E/G672E /Polr3b +/R103H double mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease. Electronic supplementary material The online version of this article (10.1186/s13041-019-0479-7) contains supplementary material, which is available to authorized users.

Defective myelination in an RNA polymerase III mutant leukodystrophic mouse

“…Previous efforts to develop a mouse model of Pol III-related leukodystrophy have been unsuccessful, either because a known homozygous disease-causing Polr3a G672E mutation had no phenotype in mice or because homozygosity for one mutation (Polr3b R103H) from a compound heterozygous variant was embryonic lethal (22,23). We reasoned that enhancing the defect of the Polr3a G672E mutation might produce a viable disease model.…”

“…Homozygosity for either mutation causes Pol III-related leukodystrophy in humans (8). However, mice that are homozygous or hemizygous for the G672E mutation alone have no apparent phenotype (22,23). The corresponding mutation in the homologous yeast subunit also had no phenotype (24).…”

POLR3-Related Leukodystrophy: Exploring Potential Therapeutic Approaches

“…POLR3 is responsible for the transcription of several non-coding RNAs (nc-RNAs) which have significant roles in translation and gene expression programs, including transfer RNAs (tRNAs), 5S ribosomal RNA, 7SL and 7SK RNAs, some microRNAs, vault RNAs, and a variety of small nucleolar RNAs, including U6 snRNA (Dieci et al, 2007 , 2013 ; White, 2011 ; Wu et al, 2012 ; Lesniewska and Boguta, 2017 ). As the genes associated with POLR3-HLD have been discovered relatively recently and attempts at generating an animal model were predominantly unsuccessful (Choquet et al, 2017 , 2019b ), the cellular and molecular mechanisms underlying the white matter pathology of this disease are largely unknown. Research is ongoing regarding the investigation of the pathophysiology of POLR3-HLD; recent modeling of the disease has been accomplished in yeast (Moir et al, 2020 ), as well as in a conditional mouse model (pre-print data, not yet peer-reviewed; Merheb et al, 2020 ).…”

RNA polymerase III transcription as a disease factor

“…Indeed, although BC1, whose levels were not affected in the mouse model, is considered the functional homolog of the primate specific BC200, the two genes have different evolutionary origins and are likely to differ in their cellular functions. A second mouse model carrying the POLR3B leukodystrophy-mediating mutation p.R103H, which led to impaired Pol III assembly in human cultured cell lines, was embryonic lethal in homozygotes (Choquet et al 2019b).…”