Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits

Abstract

Eukaryotic DNA-dependent RNA polymerases (Pols I–III) encode two distinct alpha-like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H leukodystrophy, and primary ovarian sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a “humanized” yeast that we used to characterize the molecular consequence of the TCS-causingPOLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.

Keywords:

• RNA polymerase
• alpha subunits
• Rpb11
• AC19
• POLR2J2
• POLR1D
• homologs

ACKNOWLEDGEMENTS

We thank Knutson laboratory members for their critical review of the manuscript.

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.218607.

DATA AVAILABILITY STATEMENT

The authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials. Uncropped protein gel and Western blot images are shown in Supplemental Fig. S3 and can be accessed from Mendeley data repository through accessing the link https://data.mendeley.com/datasets/848yvpr3zj.

Additional information

Funding

The work was supported by grants to B.A.K. from the U.S. National Institutes of Health [NIDCR R03-DE027785 and NIGMS R01-GM141033]. H.G.P was partially supported by funds from the Post-Baccalaureate Research Education Program at SUNY-Upstate (PREP-Up).