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. 2019 Dec 24;21(1):135.
doi: 10.3390/ijms21010135.

The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4

Sergey A Proshkin  1   2 , Elena K Shematorova  1 , George V Shpakovski  1
Affiliations

The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4

Sergey A Proshkin et al. Int J Mol Sci. .

Abstract

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3-ATF4 (not hRPB11a-ATF4) and hRpb11bα-ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3-Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11-ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

Keywords: ATF4; RNA polymerase II; hRPB11a; hRPB11bα; human isoforms of Rpb11; yeast two-hybrid system.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
(A) Two-hybrid analysis of hRPB11bα interactions. Serial dilutions of SKY191 yeast cells containing the indicated plasmid combinations were spotted onto SD-Gal/Raff plates lacking triptophan, histidine, and uracil (-WHU) to verify that yeast contains both the bait and prey plasmids (left panel) or triptophan, histidine, uracil, and leucine (-WHUL) for demonstration of the interaction between the bait and prey (central panel). Three independent clones of each of the SKY191 transformants were spotted onto X-gal containing plate (right panel) for confirmation of positive interactions (the picture was made on the red background for the better visualization of both negative and positive clones). In comparison, the yeast strains containing an empty vector pJG4-5 or library-derived plasmid encoding C-terminal part of hRPB3 were used as negative or positive controls, respectively. (B) Assessment of interaction of hRPB11bα and ATF4. SKY191 yeast cells were cotransformed with the indicated constructs and assayed for β-galactosidase activity in the liquid medium by means of the Miller method [31]. The pairs of hRPB11bα–hRPB3 and hRPB11a–hRPB3 interacting proteins were taken as a positive control. Error bars: Standard deviation; statistical analysis: * p < 0.05 for all measurements.
Figure 1
Figure 1
(A) Two-hybrid analysis of hRPB11bα interactions. Serial dilutions of SKY191 yeast cells containing the indicated plasmid combinations were spotted onto SD-Gal/Raff plates lacking triptophan, histidine, and uracil (-WHU) to verify that yeast contains both the bait and prey plasmids (left panel) or triptophan, histidine, uracil, and leucine (-WHUL) for demonstration of the interaction between the bait and prey (central panel). Three independent clones of each of the SKY191 transformants were spotted onto X-gal containing plate (right panel) for confirmation of positive interactions (the picture was made on the red background for the better visualization of both negative and positive clones). In comparison, the yeast strains containing an empty vector pJG4-5 or library-derived plasmid encoding C-terminal part of hRPB3 were used as negative or positive controls, respectively. (B) Assessment of interaction of hRPB11bα and ATF4. SKY191 yeast cells were cotransformed with the indicated constructs and assayed for β-galactosidase activity in the liquid medium by means of the Miller method [31]. The pairs of hRPB11bα–hRPB3 and hRPB11a–hRPB3 interacting proteins were taken as a positive control. Error bars: Standard deviation; statistical analysis: * p < 0.05 for all measurements.
Figure 2
Figure 2
Co-precipitation of hRPB11bα and hRPB11a isoforms of RNA polymerase II subunit POLR2J (hRPB11) with ATF4 after their heterologous expression in E. coli cells (in vitro interaction of hRPB11bα and ATF4 proteins). Fifteen percent SDS-PAGE analysis of the proteins retained on TALON agarose. Recombinant proteins 6His-hRPB11bα (lane 4) and 6His-hRPB11a (lane 5) were immobilized on TALON agarose. Untagged ATF4ΔN protein retains on the agarose if it binds immobilized proteins. MW, molecular weight marker. The gel was visualized by Coomassie Blue R250 staining as described in Materials and Methods.
Figure 3
Figure 3
Schematic representation of the uncovered protein-protein interaction: The minor subunit of Homo sapiens RNA polymerase II hRPB11bα specifically interacts with transcription factor ATF4 through its C-terminal b-Zip domain.
Figure 4
Figure 4
hRPB11bα is involved in ATF4-mediated transcription. HeLa cells were transiently transfected with 0.2 µg of the 3×ATF4-Luc reporter and 1 µg of the indicated expression vectors. Intensity of the light (560 nm) emitted as a result of the luciferase catalyzed chemiluminescent reaction is represented along the ordinate axis in relative light units (RLU = light emission/second). The data are presented as mean ± SD and representative of three independent experiments (** p < 0.005 for both measurements) performed in duplicate after normalization for transfection efficiency.
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