Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation

  1. Zhanwu Zhu
  2. Jingjing Liu
  3. Huan Feng
  4. Yanning Zhang
  5. Ruiqi Huang
  6. Qiaochu Pan
  7. Jing Nan
  8. Ruidong Miao
  9. Bo Cheng

  • Curated by eLife

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    Evaluation Summary:

    This study identifies two distinct nuclear export elements and a strong cytoplasmic anchoring sequence that restrict transcription factor GDOWN1 to the cytoplasm in normal conditions. The authors identify stress conditions that override this normal control to promote GDOWN1 nuclear localization as part of a protective response. This study will be of interest to the transcriptional regulation field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Dynamic regulation of transcription is crucial for the cellular responses to various environmental or developmental cues. Gdown1 is a ubiquitously expressed, RNA polymerase II (Pol II) interacting protein, essential for the embryonic development of metazoan. It tightly binds Pol II in vitro and competitively blocks the binding of TFIIF and possibly other transcriptional regulatory factors, yet its cellular functions and regulatory circuits remain unclear. Here, we show that human GDOWN1 strictly localizes in the cytoplasm of various types of somatic cells and exhibits a potent resistance to the imposed driving force for its nuclear localization. Combined with the genetic and microscope-based approaches, two types of the functionally coupled and evolutionally conserved localization regulatory motifs are identified, including the CRM1-dependent nucleus export signal (NES) and a novel Cytoplasmic Anchoring Signal (CAS) that mediates its retention outside of the nuclear pore complexes (NPC). Mutagenesis of CAS alleviates GDOWN1’s cytoplasmic retention, thus unlocks its nucleocytoplasmic shuttling properties, and the increased nuclear import and accumulation of GDOWN1 results in a drastic reduction of both Pol II and its associated global transcription levels. Importantly, the nuclear translocation of GDOWN1 occurs in response to the oxidative stresses, and the ablation of GDOWN1 significantly weakens the cellular tolerance. Collectively, our work uncovers the molecular basis of GDOWN1’s subcellular localization and a novel cellular strategy of modulating global transcription and stress-adaptation via controlling the nuclear translocation of GDOWN1.

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  1. Version published to 10.7554/elife.79116 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    1&2) Throughout the paper, the authors use a BiFC assay to monitor direct interactions between GDOWN1 and other transcription factors in the cell. While this assay works well for their experiments, we are unsure why GDOWN1 appears to interact with every protein found in the cytoplasm. This is particularly concerning when we look at GDOWN1 interacting with itself (Figure 1D), as GDOWN1 is not known to self-oligomerize. The authors should provide a negative control that GDOWN1 does not non-specifically interact with any cytoplasm-localized protein. Additionally, every GDOWN1 truncation tested was able to interact with NELF-E. We are unsure why each truncation tested (given that they tested multiple non-overlapping GDOWN1 regions) can interact with NELF-E. Do the authors believe that NELF-E directly interacts with every tested GDOWN1 construct? We believe that demonstration of BiFC specificity is critical for the conclusions drawn in the manuscript.

    Thank you for your comments and valuable suggestions! We added more negative BiFC controls in the revised manuscript to demonstrate the specificity of BiFC assays (Figure 1——figure supplement 1D). Since both reviewers brought up this question, we provided our answers to this question above in the “Common concerns by the Reviewers” session (Q#1).

    1. The authors note that the NES1 site is not as strong as the NES2 site at regulating exportin 1-dependent nuclear export. However, they suggest this is because mutating the NES2 site is more likely to disrupt the CAS site nearby. We ask the authors to expand on this concept. Do they have direct evidence that NES2 disrupts CAS activity (such as regulating its association with the nuclear pore complex)?

    From Figure 4A, we can see that both NES1 (4A-b) and NES2 (4A-d) work as functional nuclear export signals. When NES1 was mutated (4A-c), NES2 and CAS both remained functional in blocking GDOWN1’s nuclear shuttling upon LMB addition. However, when NES2 was mutated (4A-e), comparing the localization changes before and after LMB addition, we concluded that NES1 remained functional, while the cytoplasmic retention activity of CAS was partially lost. From the quantification of the images, it seems that NES1 has a stronger activity than NES2 in terms of the LMB responsiveness/CRM1-depentent nuclear export activity, while apparently NES2 exhibits another layer of regulation/correlation on the CAS activity.

    To further confirm this observation, we generated a HeLa stably cell line expressing GDOWN1(NES2 mutant)-Venus and tested the subcellular localization of this mutant. As shown in the Figure 4C of the revised manuscript, compared with the wild type GDOWN1, loss of the NES2 activity directly caused the loss of the perinuclear staining, which was consistent to the defect of the CAS mutant. These results further support that the mutagenesis of NES2 disrupts the CAS-mediated association to the nuclear pore complex.

    1. The authors show the critical role of the NES1, NES2, and CAS sites for the localization and function of GDOWN1. Have the authors checked post-translational modification databases to check if any of the identified sites could be post-translationally modified and thereby regulated? Elucidation of the mechanism by which GDOWN1 localization is regulated is of broad interest to the transcription community.

    Good suggestion! It is worthy of checking and testing the potential modifications on the key arginines identified in CAS (R352, R354, and R357). We did check the web tools for arginine methylation site prediction (http://msp.biocuckoo.org/online.php), but none pf the known motifs was found to match with the CAS sequences of GDOWN1. In addition, our pilot studies for the treatments using the inhibitors of arginine methyltransferases (- or + LMB) did not result in any nuclear accumulation of GDOWN1 (data not shown). So far, we do not have any strong evidence to confirm that these arginines are directly modified in our assays, and we cannot exclude the possibilities of other amino acids nearby also play key roles on the CAS function. Thus, more research is badly needed to uncover the regulatory mechanism of CAS.

  3. eLife

    Evaluation Summary:

    This study identifies two distinct nuclear export elements and a strong cytoplasmic anchoring sequence that restrict transcription factor GDOWN1 to the cytoplasm in normal conditions. The authors identify stress conditions that override this normal control to promote GDOWN1 nuclear localization as part of a protective response. This study will be of interest to the transcriptional regulation field.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The exact role of Gdown1 has remained something of an enigma. At the biochemical level, Gdown1 can be strongly inhibitory to both preinitiation complex assembly and transcript elongation. However, there are now many examples, including those shown here, where the loss of Gdown1 can have very little effect. Other work has shown that Gdown1 is typically cytoplasmic, focusing attention on the mechanisms that keep Gdown1 out of the nucleus. Here the authors conclusively demonstrate that Gdown1 has two independent protein domains that drive nuclear export and a separate domain that normally anchors Gdown1 on the cytoplasmic face of the nucleus. When these localization domains are mutated and Gdown1 has routine nuclear access transcription is inhibited, apparently in part because of reduced Pol II levels. This in turn compromises cell viability. Cell viability can also be reduced by exposure to arsenite. In that case, Gdown1 gains nuclear access and reduces the negative effect, presumably because temporary transcriptional shutdown is protective in that case.

  5. eLife

    Reviewer #2 (Public Review):

    Zhu et al. studied GDOWN1, a known RNA Polymerase II transcriptional regulator, and specifically investigated regulation of its localization. Using fluorescent microscopy and pull-down assays, the authors determined the multi-valent nature by which GDOWN1 constitutively remains in the cytoplasm. They found three non-redundant regulator domains within GDOWN1 that ensure GDOWN1 remains in the cytoplasm and only localizes into the nucleus upon stress. Specifically, the authors found two independent domains that directly bind to exportin 1, which ensures the proper export out of the nucleus. Further, they find a third domain that directly interacts with the Nuclear Pore Complex. These three domains regulate nuclear import/export. Additionally, they demonstrate that GDOWN1 inhibits transcriptional activity upon nuclear localization. Their data suggests GDOWN1 is a transcriptional regulator that regulates transcription during cell-stress.

    Overall, this is a solid paper, the data supports the authors' claims, and only a few points require clarification.

    1. Throughout the paper, the authors use a BiFC assay to monitor direct interactions between GDOWN1 and other transcription factors in the cell. While this assay works well for their experiments, we are unsure why GDOWN1 appears to interact with every protein found in the cytoplasm. This is particularly concerning when we look at GDOWN1 interacting with itself (Figure 1D), as GDOWN1 is not known to self-oligomerize. The authors should provide a negative control that GDOWN1 does not non-specifically interact with any cytoplasm-localized protein.
    2. Additionally, every GDOWN1 truncation tested was able to interact with NELF-E. We are unsure why each truncation tested (given that they tested multiple non-overlapping GDOWN1 regions) can interact with NELF-E. Do the authors believe that NELF-E directly interacts with every tested GDOWN1 construct? We believe that demonstration of BiFC specificity is critical for the conclusions drawn in the manuscript.
    3. The authors note that the NES1 site is not as strong as the NES2 site at regulating exportin 1-dependent nuclear export. However, they suggest this is because mutating the NES2 site is more likely to disrupt the CAS site nearby. We ask the authors to expand on this concept. Do they have direct evidence that NES2 disrupts CAS activity (such as regulating its association with the nuclear pore complex)?
    4. The authors show the critical role of the NES1, NES2, and CAS sites for the localization and function of GDOWN1. Have the authors checked post-translational modification databases to check if any of the identified sites could be post-translationally modified and thereby regulated? Elucidation of the mechanism by which GDOWN1 localization is regulated is of broad interest to the transcription community.

  6. Version published to 10.1101/2022.03.29.486178v1 on bioRxiv