Clinical and Transcriptomic Characterization of a Polr3a Knock-in Mouse

Karine Choquet1,2,3, Roxanne Larivière1, Marie-Josée Dicaire1, Claudia L. Kleinman2,3, Bernard Brais1,2

1. Neurogenetics of Motion Laboratory, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Canada; 2. Department of Human Genetics, McGill University, Montreal, Canada; 3. Lady Davis Institute, Jewish General Hospital, Montreal, Canada

Background: RNA Polymerase III (Pol III) is an essential enzyme responsible for the transcription of transfer RNAs (tRNAs) and many non-coding RNAs. Mutations in POLR3A, encoding the largest subunit of Pol III, cause Pol III-related hypomyelinating leukodystrophy (HML), characterized by hypomyelination, cerebellar atrophy and variable extra-neurological involvement. Impaired coordination, balance and locomotion are common features among Pol III-related HML patients. Interestingly, the majority of French Canadian cases are homozygous for the founder POLR3A c.2015G>A (p.G672E) mutation.

Hypothesis and Objectives: We hypothesized that mutations in POLR3A alter Pol III’s transcription efficiency, leading to deregulation of key Pol III transcripts essential for normal neuronal and glial development and survival. To identify the transcripts responsible for the pathological phenotype and to unravel the pathophysiological mechanisms of the disease, we generated a knock-in (KI) mouse model homozygous for the Polr3a G672E mutation. Here, we report on the initial phenotypic characterization of this mouse model and the first transcriptomic analysis of its central nervous system (CNS).

Methods and Results: We found that homozygous Polr3a KI mice are viable, fertile and do not manifest a gross motor phenotype before the age of 8 months. To determine if they have a subtler motor phenotype, we submitted WT and KI mice to the rotarod and the open field test at P55 as a first time point in an ongoing trial. At this age, the KI mice have mildly impaired coordination and general locomotion compared to WT mice. In parallel, since accurate quantification of Pol III transcripts is challenging because of their strong secondary structures and repetitive sequences, we developed a customized RNA-Sequencing and analysis strategy to compare expression levels of Pol III transcripts. We first performed a pilot experiment on whole brains of two WT and two KI mice at P30. Our results suggest that accurate quantification of Pol III transcripts is possible using our customized strategy. In this pilot study, we found no differences in expression levels of Pol III transcripts between WT and KI, likely due to the heterogeneity of whole brain tissue and the time point studied. These preliminary results confirm that if there is a time in development, a CNS structure and/or a cell population that have impaired expression levels of Pol III transcripts, we have strategies to quantify these differences and to study their implications for the pathophysiology of Pol III-related HML.