POLR3B encodes the second-largest catalytic subunit of RNA polymerase III (Pol III), a DNA-dependent RNA polymerase essential for transcribing small non-coding RNAs including 5S rRNA, tRNAs, snRNAs, and miRNAs from approximately 500 genomic loci 1. As part of the Pol III active center, POLR3B coordinates a magnesium ion and provides lysine residues facilitating Watson-Crick base pairing during nucleotide addition to the nascent RNA 2. Beyond transcription, Pol III functions as a nuclear and cytosolic DNA sensor in innate immunity, detecting non-self DNA and triggering type I interferon responses through the RIG-I pathway [UniProt annotation supported by 38]. Biallelic POLR3B mutations cause 4H leukodystrophy (hypomyelination, hypodontia, hypogonadotropic hypogonadism), characterized by motor delay before age 6 years, with a milder disease course than POLR3A mutations 4. Pathogenic variants impair oligodendrocyte precursor proliferation and differentiation, reducing mature oligodendrocyte numbers during myelinogenesis 5. De novo heterozygous POLR3B variants cause developmental and epileptic encephalopathy with myoclonic-atonic seizures, ataxia, microcephaly, and developmental delay, typically presenting between 6 months and 4 years 6. Rare de novo mutations also associate with early-onset demyelinating Charcot-Marie-Tooth disease 7. POLR3B dysfunction leads to decreased Pol III transcript levels, altered small ncRNA profiles, and can be detected via tRNA fragment biomarkers 8.