Gpn2 and Rba50 Directly Participate in the Assembly of the Rpb3 Subcomplex in the Biogenesis of RNA Polymerase II

Abstract

RNA polymerase II (RNAPII) is one of the central enzymes in cell growth and organizational development. It is a large macromolecular complex consisting of 12 subunits. Relative to the clear definition of RNAPII structure and biological function, the molecular mechanism of how RNAPII is assembled is poorly understood, and thus the key assembly factors acting for the assembly of RNAPII remain elusive. In this study, we identified two factors, Gpn2 and Rba50, that directly participate in the assembly of RNAPII. Gpn2 and Rba50 were demonstrated to interact with Rpb12 and Rpb3, respectively. An interaction between Gpn2 and Rba50 was also demonstrated. When Gpn2 and Rba50 are functionally defective, the assembly of the Rpb3 subcomplex is disrupted, leading to defects in the assembly of RNAPII. Based on these results, we conclude that Gpn2 and Rba50 directly participate in the assembly of the Rpb3 subcomplex and subsequently the biogenesis of RNAPII.

Keywords: Gpn2; RNA polymerase II; Rba50; assembly factor.

FIG 1

RPB12 and RBA50 suppress the gpn2^ts mutant. (A) Alignment of the Gpn2 amino acid sequences of the yeast (S. cerevisiae and Schizosaccharomyces pombe) and human (Homo sapiens) proteins. Gpn2 amino acid sequences of the budding yeast, fission yeast, and human proteins were aligned by ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/) using the default settings. The conserved G domains and the mutation sites of gpn2^ts are indicated. (B) Growth of wt (YFZ28) and gpn2^ts (YFZ 29) cells on YPD plates at different temperatures and sensitivity of gpn2^ts cells to 25 μg/μl MPA. (C) Exponentially growing wt (YFZ28) and gpn2^ts (YFZ 29) cells at 25°C were shifted to 34°C. Samples were taken at the indicated time points. TCA-processed whole-cell extracts were analyzed by immunoblotting with anti-myc (GPN2) antibodies. A Ponceau S-stained region of the same membrane as that used for immunoblotting is shown as a loading control. (D) Multicopy suppressors of gpn2^ts identified in this screening. “Hit” indicates the number of clones obtained. (E) Fivefold serial dilutions of exponentially growing wt cells (YFZ28) carrying the pRS425 mock plasmid and the gpn2^ts mutant (YFZ29) carrying the indicated constructs were spotted onto SD-Leu plates with or without MPA and incubated at the indicated temperatures for 3 days. (F) RPB12 or RBA50 corrects the nuclear localization of RNAPII in the gpn2^ts cells. Wild-type cells (YFZ50) carrying empty plasmids (pRS425) and the gpn2^ts mutant (YFZ55) carrying the indicated constructs were grown to mid-exponential phase at 25°C, shifted to 32°C for 2 h, and then fixed for Rpb1-GFP distribution analysis by fluorescence microscopy. The arrows indicate the cytoplasmic foci of Rpb1-GFP in the gpn2^ts mutant at 32°C.

FIG 2

Gpn2 is required for the assembly of RNAPII at an early step. (A) Exponentially growing YFZ75 (Rpb3-3HA Gpn2-13myc Rpb1-GFP) and control YFZ50 (Gpn2-13myc Rpb1-GFP) cells, untreated or treated with 2 μg/ml α-amanitin for 1 h at 30°C, were collected and subjected to coimmunoprecipitation (Co-IP) using anti-HA beads (Rpb3). The coimmunoprecipitated proteins were analyzed by immunoblotting with either anti-GFP (Rpb1), anti-myc (Gpn2), or anti-HA (Rpb3) antibodies. The right panel shows the quantification of normalized Rpb1 and Gpn2 with Rpb3 immunoprecipitation with/without α-amanitin treatment. (B) Cell extracts from exponentially growing YFZ50 (Rpb1-GFP Gpn2-13myc) and the untagged control YFZ44 (Rpb1-GFP) were immunoprecipitated using anti-myc beads (Gpn2). The immunoprecipitated proteins were analyzed by immunoblotting with either anti-GFP (Rpb1) or anti-myc (Gpn2) antibodies. (C) Exponentially growing cultures of wt (YFZ75, Rpb1-GFP Gpn2-13myc Rpb3-3HA) and gpn2^ts (YFZ90, Rpb1-GFP Gpn2^ts-13myc Rpb3-3HA) cells as well as the mock control (YFZ50, Rpb1-GFP Gpn2-13myc) at 25°C were shifted to 36.5°C for 1.5 h and harvested. Rpb3 was immunopurified with anti-HA beads from the indicated extracts. Copurified Rpb1 and Gpn2 were analyzed with anti-GFP and anti-myc antibodies, respectively. (D) The copurified proteins from the sample in panel C were separated by SDS-PAGE and stained with Coomassie blue, followed by detection by mass spectrometry. The normalized quantification of copurified Rpb1 and Rpb2 is shown on the right-side panel. (E) Exponentially growing YFZ94 (Gpn2-mCherry Rpb3-GFP) cells were fixed and visualized on a Delta Vision Elite microscope.

FIG 3

Gpn2 targets Rpb12 for the assembly of the Rpb3 subcomplex. (A) Two-hybrid interactions were judged by spot tests on two types of dropout (DO) plates with the corresponding proteins in human cells: high-stringency 4DO (SD-adenine, -histidine, -leucine, and -tryptophan) and the control 2DO (SD-leucine and -tryptophan). Reciprocal combinations of fusions with the GAL4 DNA-binding domain (BD) to hGpn2 and the GAL4 activation domain (AD) to each component of core human RNAPII were examined (the stalk part Rpb4/7 was not shown). (B) Coomassie blue-stained SDS-PAGE showing Escherichia coli purified recombinant GST-Rpb12 and GST. (C) Recombinant GST-Rpb12 or GST was used as bait in a pulldown assay with whole-cell extracts from exponentially growing wt cells (YFZ75). Proteins pulled down in the assay were analyzed by immunoblotting with anti-myc (Gpn2) or anti-HA (Rpb3) antibodies (1/50 lysate used in each pulldown was loaded as input control). (D) The genetic rescue assay of the indicated genes was examined following the assay described for Fig. 1E. (E) The same Co-IP experiment as that described for Fig. 2C was performed, and the copurified proteins were probed using antibodies against the indicated proteins.

FIG 4

Rba50 is an essential assembly factor for the formation of the Rpb3 subcomplex. (A) Growth of wt (YFZ69) and rba50-3 (YFZ77) on YPD plates at 24°C and 37°C. (B) Equal numbers of GPN2-GFP, rba50-3, or double mutant cells were grown at 28°C for 8.5 h. The data for the graph of the growth curves were the means for three replicates. OD₆₀₀, optical density at 600 nm. (C) Exponentially growing YFZ50 (Gpn2-13myc Rpb1-GFP) and YFZ74 (Rba50-3HA Gpn2-13myc Rpb1-GFP) cells were collected and subjected to Co-IP using anti-HA beads (Rba50). The coimmunoprecipitated proteins were analyzed by immunoblotting with either anti-GFP (Rpb1), anti-myc (Gpn2), or anti-HA (Rpb3) antibodies. (D) Two-hybrid interaction of human GPN2 and RPAP1 (the homolog of yeast Rba50). The procedure of the two-hybrid assay is the same as described for Fig. 3D. (E) Wild-type cells (YFZ44) and rba50-3 mutants (YFZ96) expressing Rpb1-GFP were grown to mid-exponential phase at 24°C, shifted to 34°C for 2 h, and then fixed for Rpb1-GFP distribution analysis by fluorescence microscopy. (F) Distributions of Gpn2-mCherry and Rba50-GFP. Exponentially growing YFZ95 cells (Gpn2-mCherry Rba50-GFP) were fixed, and their fluorescence was visualized by fluorescence microscopy. (G) Fivefold serial dilutions of exponentially growing wt cells (YFZ28) carrying the pRS425 mock plasmid and the rba50-3 mutant (YFZ77) carrying the indicated constructs were spotted onto SD-Leu⁻ plates with/without MPA and incubated at the indicated temperatures for 3 days. (H) Wild-type cells (YFZ87, Rpb3-GFP Rba50-13myc), rba50-3 mutant cells (YFZ97, Rpb3-GFP Rba50^ts-13myc), and mock cells (YFZ73, Rpb3-3HA Rba50-13myc) were grown to log phase at 30°C and then shifted to 36.5°C for an additional 1 h of incubation. The cells were harvested and subjected to the preparation of cell extracts and subsequent immunoprecipitation. Rpb3 and Rpb3-associated proteins were brought down with GFP-Trap beads. Rba50, Rpb11, and Rpb12 were detected by Western blotting with corresponding antibodies.

FIG 5

A working model of Gpn2 and Rba50 acting for the assembly of the Rpb3 subcomplex. Shown is a model for the recruitment of Rpb12 to Rpb3 in a Gpn2- and Rba50-coordinated manner. The Rpb3 subcomplex is followed by the Rpb2 subcomplex, finishing with the addition of the Rpb1 subcomplex.