Xue, J.; Jiang, Z.; Zhang, J.; Li, C.

Spliceosomal small nuclear RNA (snRNA) genes show unusually high germline mutation rates, with R-loop formation previously implicated in this mutability. Our cross-species rare-variant and de novo mutation analyses show that hypermutability is concentrated in RNA polymerase II-transcribed spliceosomal snRNA genes, with strong enrichment in primates but a much weaker excess in rodents and chicken, consistent with lineage differences in APOBEC3 repertoires. Human Pol II-transcribed spliceosomal snRNA genes show mutational strand asymmetry, APOBEC3-associated mutational signatures and strong APOBEC3B binding signals. R-loops are also frequent at these loci, including in mouse genes with much weaker hypermutability, indicating that R-loop formation alone is insufficient to explain the phenotype. Together, cross-species comparisons support lineage-specific APOBEC3 activity, rather than R-loop formation alone, as a major contributor to spliceosomal snRNA gene hypermutability.