Humanization of the  rpb9  Locus in Fission Yeast Reveals Conserved and Divergent Roles of  rpb9  and Human  POLR2I .
 Background/Objectives : RNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in humans is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I functions is inferred from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the  rpb9  gene of the fission yeast  Schizosaccharomyces pombe  to examine the functional capabilities of POLR2I.  Methods : We edited the genomic  rpb9  locus in  S. pombe  so that it encodes the human POLR2I protein, and investigated functional and structural conservation.  Results : With our humanized yeast system, we find widespread functional complementation by human  POLR2I  of  S. pombe rpb9  roles in yeast growth, chronological aging, and stress responses. We also find that  POLR2I  complements novel roles for yeast  rpb9  in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that  POLR2I  cannot complement the role of  rpb9  in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly,  POLR2I  could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins.  Conclusions : Our study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast  rpb9  and its human homolog,  POLR2I . In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of  rpb9  in heterochromatin formation and chemoresistance.
Finkel JM, Williams MG, Nirmal MB, Pandey S, Howe ED, Liu CT, Lohman JR, Sharma N, Vo TV