Understanding the molecular basis of the mutation in the RNA polymerase III subunit Rpc10 (R41W)- associated with hypomyelinating leukodystrophy in the yeast homolog Rpc11.

RNA Polymerase III (RNAP III) is crucial for synthesizing abundant non-coding RNAs like tRNAs and 5S rRNA. Its activity is tightly controlled, and disruptions often lead to severe diseases. Mutations in RNAP III subunits are linked to a range of human disorders, including hypomyelinating leukodystrophy (HLD). Among the various mutations identified in patients with HLD, the POLR3K (RPC10)-R41W mutation has drawn particular attention because of its localization in the linker domain of RPC10, a domain that connects the N-terminal (NTD) and C-terminal (CTD) zinc ribbon motifs. However, the mechanism by which the RPC10-R41W mutation contributes to the disease pathogenesis remains poorly understood. This study modeled the human POLR3K-R41W (RPC10-R41W) mutation in the yeast homolog C11 at the conserved position (R43W in S. cerevisiae and R41W in S. pombe). We systematically investigated its impact on RNAP III functions, including RNA 3'-end cleavage, transcription termination, and overall cell growth. Our findings demonstrate that this mutation specifically impairs the RNA 3' cleavage activity of C11 without compromising transcription termination. We observed slow growth in yeast models, particularly under lower temperatures, suggesting a potential stress response mechanism. Additionally, the mutation was associated with the lengthening of 3'-oligo(U) sequences in tRNAs. This C11 mutation also shows a fatal phenotype in the Sen1-deleted strain, an important helicase for RNAP III transcription termination in S. pombe. These observations provide valuable insight into how this linker domain mutation affects the cleavage activity, potentially contributing to cellular stress responses and the broader implications for HLD.