bioRxiv2026
Pathogenic variants in EXOSC3, a noncatalytic subunit of the RNA exosome, cause pontocerebellar hypoplasia type 1B (PCH1B), yet substantial variability in disease severity is observed among individuals carrying different EXOSC3 alleles. The molecular mechanisms of RNA exosome dysfunction in individuals carrying EXOSC3 p.G191 variants remains unresolved. To address this, we generated CRISPR/Cas9-engineered human cell models harboring EXOSC3 p.G191 variants and performed integrated transcriptomic, proteomic, and computational structural analyses. EXOSC3 p.G191 variants produced extensive, allele- and dosage-dependent alterations in gene expression and splicing, with heterozygous variants causing broad but attenuated disruption relative to homozygous EXOSC3 G191D/G191D cells. All EXOSC3 G191 variants promoted increased skipping of exon 3 in EXOSC3 transcripts, generating a short isoform predicted to encode an unstable proteoform. Molecular dynamics and {lambda}-dynamics simulations predicted substantial thermodynamic destabilization of all EXOSC3 G191 variant proteoforms, consistent with reduced protein abundance and thermal stability measured by global proteomics and PISA. At the protein complex level, EXOSC3 p.G191 variants were associated with coordinated decreases in all RNA exosome core subunits and the exonuclease EXOSC10, consistent with destabilization of RNA exosome assembly and orphan protein decay. In contrast, the catalytic exonuclease DIS3 and multiple independent RNA processing pathways were upregulated, indicating compensatory recalibration of RNA metabolism. Together, these findings link variant-induced alternative splicing, RNA exosome complex destabilization, and adaptive network responses to phenotypic variability in EXOSC3 p.G191-associated disease.