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: AC19; POLR1D; POLR2J2; RNA polymerase; Rpb11; alpha subunits; homologs.

FIG 1

Conservation of the alpha-like subunits in yeast and human. (A) Common names for small and large ⍺-like heterodimer subunits. y, yeast; h, humans. (B) Percent identity between small alpha-like subunit protein homologs. (C) Alignment of yeast and human small alpha-like subunit homologs protein sequences. Residue conservation between homologs is indicated with purple highlighting. Light purple is conserved between two homologs and it ranges up to dark purple where the residue is conserved between all four homologs. Red bar indicates the boundaries of the alpha-motif. An asterisk denotes residues mutated to alanine in the alpha-motif mutant (Δ⍺m), and the green bar denotes residues deleted in the C-terminal helix deletion mutant (ΔCTD). Dashes above the protein sequence indicate every 10 residues. (D) Surface representation of the human RNA polymerase I structure (PDB 7OB9). The highlighted box is a zoomed in ribbon structure of the alpha–like heterodimer. POLR1C and POLR1D are colored in blue and purple, respectively. Residues mutated in the Δ⍺m mutant are colored in red, and the residues deleted in the ΔCTD mutant are colored in green.

FIG 2

Effect of alpha-motif and C-terminal helix mutations on yeast Rpb11 and its human ortholog POLR2J2. (A) In vitro Ni-NTA pulldown of yeast His₆-tagged Rpb11 variants and HA-tagged Rpb3 co-expressed in bacteria. SDS-PAGE analysis and Coomassie stain of Ni-NTA purified proteins and inputs are shown. (B) Yeast plasmid shuffle assay of Rpb11 variants grown on glucose media containing 5-FOA. (C) Western blot analysis assessing protein expression levels of yeast Rpb11 variants. (D) Western blot analysis of immunopurified HA-tagged Rpb11 variants in a FLAG-tagged Rpb3 yeast strain. Input of the immunopurified proteins is shown. (E) In vitro Ni-NTA pulldown of human His₆-tagged POLR2J2 variants and HA-tagged POLR2C co-expressed in bacteria. Western blot analysis of Ni-NTA purified proteins and SDS-PAGE analysis and Coomassie staining of Ni-NTA pulldown inputs are shown.

FIG 3

Effect of alpha-motif and C-terminal helix mutations on the yeast AC19 and its human ortholog POLR1D. (A) In vitro Ni-NTA pulldown of yeast His₆-tagged AC19 variants and HA-tagged AC40 co-expressed in bacteria. SDS-PAGE analysis and Coomassie stain of Ni-NTA purified proteins and inputs are shown. (B) Yeast plasmid shuffle assay of AC19 variants grown on glucose media containing 5-FOA. (C) Protein expression levels of AC19 variants in yeast analyzed by Western blot. (D) Western blot analysis of immunopurified HA-tagged AC19 variants in a FLAG-tagged AC40 yeast strain. Input of the immunopurified proteins is shown. (E) In vitro Ni-NTA pulldown of human His₆-tagged POLR1D variants and HA-tagged POLR1C co-expressed in bacteria. Western blot analysis of Ni-NTA purified proteins and SDS-PAGE analysis and Coomassie staining of Ni-NTA pulldown inputs are shown.

FIG 4

Different residues are required for yeast Rpb11 and AC19 biological function and heterodimerization. (A and B) Heat map summary of yeast plasmid shuffle growth assays of various Rpb11 (Panel A) and AC19 (Panel B) variants grown at the indicated temperatures. “WT,” wild-type; “EV,” empty vector. Boxes with light coloring indicates wild-type-like growth while dark coloring indicates a lethal mutation. The bar graph to the right of the heat map shows the protein expression relative to respective wild-type proteins. (C to F) Surface representation of Rpb11 (PDB 1WCM) (Panels C and D) and AC19 (PDB 4C2M) (Panels E and F). Residues classified as Group 1 (growth defect and stable protein) are colored in red. Group 2 residues (growth defect and unstable protein) are colored in yellow, and Group 3 residues (no growth defect) are colored in gray. The classification of conservative mutations are depicted in Panels C and E, and radical mutations are depicted in Panels D and F. (G) Western blot analysis of immunopurified HA-tagged Rpb11 variants in a FLAG-tagged Rpb3 yeast strain and inputs are shown. (H) Western blot analysis of immunopurified HA-tagged AC19 variants in a FLAG-tagged AC40 yeast strain and inputs are shown.

FIG 5

Yeast-human AC19 chimera is more sensitive to regional mutations than yeast AC19. (A) Cartoon schematic of humanized AC19 chimera. (B) Ribbon structure of yeast AC19 (PDB 4C2M) made in PyMOL. The AC19 sequence is colored in pink and the region replaced with the human POLR1D sequence is colored in purple. (C and D) Yeast plasmid shuffle assay of AC19 chimera variants grown on glucose media containing 5-FOA at 30 °C (Panel C) and at 37 °C (Panel D). (E) Protein expression levels of AC19 chimera variants in yeast analyzed by Western blot.

FIG 6

Yeast-human AC19 chimera is sensitive to the clinically relevant G73E mutation associated with TCS. (A) Structural alignment of Yeast Pol I (PDB 4C2M) and Human Pol I (PDB 7OB9) ribbon structures. Human Pol I in dark gray, yeast Pol I in light gray, human POLR1C in dark blue, yeast AC40 in light blue, human POLR1D in purple, and yeast AC19 in light pink. Boxes below zoom in on the wild-type POLR1D G52 and AC19 G73 position (left box) and their mutated G52E and G73E forms (right box). Red dotted circle indicates the location of the G52 and G73 side chains. (B) Yeast plasmid shuffle assay of indicated AC19 variants grown on glucose media containing 5-FOA. (C) Protein expression levels of indicated AC19 variants in yeast analyzed by Western blot. (D) Western blot analysis of immunopurified HA-tagged AC19 variants in a FLAG-tagged AC40 yeast strain and inputs are shown. (E) Yeast plasmid shuffle assay of indicated y/h AC19 chimera variants grown on glucose media containing 5-FOA. (F) Protein expression levels of indicated y/h AC19 chimera variants in yeast analyzed by Western blot. (G) Western blot analysis of immunopurified HA-tagged y/h AC19 chimera variants in a FLAG-tagged AC40 yeast strain and inputs are shown. (H and I) Western blot analysis of immunopurified HA-tagged y/h AC19 chimera variants in a FLAG-tagged A190 (Panel H) and Rpc2 (Panel I) yeast strain and inputs are shown. (J) Yeast plasmid shuffle assay of indicated y/h AC19 chimera variants grown on glucose or galactose media containing 5-FOA.

FIG 7

Differential contribution of the small alpha-like subunits alpha-motif and CTD for heterodimerization in yeast and human. Modular schematic of the small and large alpha-like subunit heterodimer pairs in yeast and human depicting the distinct contributions of the alpha-motif (red box) and CTD (green bar) for their interaction. The small yeast and human alpha-like subunits are depicted as pink and purple bars, respectively. The large yeast and human subunits are colored in violet and blue, respectively. Arrow thickness indicates the contribution of the region to the heterodimerization (a greater contribution is shown by thicker arrows). Protein names are labeled to the left of the schematic, along with the corresponding yeast = and human cartoons next to the protein names indicating the species. The yeast-human chimera combines both yeast and human cartoons. The Pol the heterodimer is present and is indicated to the left of the species. “II” indicates Pol II, and “I/III” indicates Pols I and III.