Nirmal, M. B. | bioRxiv Science

bioRxiv2026

Humanization of the rpb9 locus in fission yeast reveals conserved and divergent roles of rpb9 and human POLR2I

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives 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. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith 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. ConclusionsOur 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.

synthetic biology↗

bioRxiv2025

Analysis of a Rpb2 mutant in Schizosaccharomyces pombe reveals a non-canonical role for Elp1 in regulating RNAi-dependent heterochromatin assembly

Heterochromatin is a repressive epigenetic state that suppresses transcription and safeguards genomic integrity. However, the full mechanism of how it is regulated remains elusive. Here, we focus on a previously described Pol II variant called rpb2-N44Y, which has a single substitution mutation within the Rpb2 subunit of Pol II that reduces RNAi-dependent heterochromatin. Through CRISPR-mediated site-directed mutagenesis, we find that rpb2-N44Y is a gain-of-function mutation. Furthermore, the heterochromatin defects of the rpb2-N44Y mutant requires a subunit of the Elongator complex called Elongator Protein 1 (Elp1), a protein that canonically promotes in mcm5s2U34 tRNA modifications. Intriguingly, we find that loss of Elp1, but not of other Elongator subunits such as Elp3, can robustly suppress heterochromatin defects in the rpb2-N44Y mutant. Elp1 acts independently of the mcm5s2 U34 tRNA modification to suppress RNAi-dependent heterochromatin at the pericentromere and the levels of small interfering RNAs (siRNAs) at affected heterochromatin. Overall, our study reveals two distinct Rpb2-centric pathways, via RNAi or Elp1, that can positively or negatively regulate heterochromatin, respectively. Furthermore, our findings reveal the first evidence of a chromatin function for Elp1 that is distinct from its canonical role in tRNA modifications. This work expands our understanding of how Elp1 can influence chromatin biology. Article summaryRNAi-dependent heterochromatin plays a key role in silencing gene expression from fission yeast to animals. However, it remains unclear what are all the factors that regulate this heterochromatin type. Here, the authors performed genetic interaction analyses to identify the conserved elp1 gene as having the potential to regulate RNAi-dependent heterochromatin. Furthermore, the authors devised a separation-of-function mutant to find that this chromatin function of elp1 is distinct from its canonical role in tRNA modifications. These findings expand our knowledge about the human disease-relevant elp1 gene beyond its well-known roles in the Elongator complex and in tRNA modifications.

genetics↗