The results presented in this paper provide insights into the function of the two Z-DNA binding domains and different isoforms of human ZBP1 and demonstrate association and interaction of ZBP1 isoforms with SGs.
All proteins that can specifically bind to left-handed Z-DNA and Z-RNA known to date contain Zα domains (15
). Although the structure of all crystallized Zα domains is remarkably similar, not all of them can actually bind the Z-form by their own (10
). Using CD spectroscopy we have shown that ZαZBP1
(encoded by exon 2) and ZβZBP1
(encoded by exon 4) can bind to left-handed Z-DNA independently of each other, resulting in comparable CD spectra of DNA. This confirms a recent report where both domains, when tested independently, converted DNA from the B- to the Z-form (37
). In addition to this finding this report shows that when both Zα domains are tethered to one another, as in the full length protein, the capacity to convert DNA from the B-form to the Z-form is greatly enhanced. Consistently, the results of EMSA experiments show that formation of DNA–protein complexes is greatly augmented when Zαβ is present in one protein molecule as compared to the situation when Zα or Zβ were tested separately or when Zα or Zβ are mixed together.
The complexes of Zα proteins and DNA exhibited different levels of mobility. The larger protein Zαβ (19 kDa) forms complexes which migrate faster and thus appear to be smaller than the complexes formed by the single Zα (9.5 kDa) or Zβ (10.9 kDa) domains. This might be caused by the binding of more protein molecules to the probe if the protein is smaller, leading to the formation of larger complexes.
The importance of two intact Zα domains in one molecule for Z-DNA binding is further supported by the results of our mutagenesis experiments. Mutation of conserved amino acids that contact Z-DNA as seen in the crystal structures (N46, Y50, N141, Y145) resulted in a dramatically reduced capacity to bind Z-DNA. Interestingly, Z-DNA binding of Zαβ was strongly affected even when these amino acids were substituted in only one of the Zα domains.
The results of our in vitro Z-DNA binding experiments led us to hypothesize that the two major isoforms of ZBP1 might show different subcellular distribution. Indeed, following transfection into HeLa cells, full length ZBP1 and ZBP1ΔZα displayed strikingly different subcellular localizations. While both isoforms displayed a predominant cytoplasmic localization, full length ZBP1 showed a diffuse finely punctate distribution, whereas ZBP1ΔZα localized in large granules. Importantly, the finely punctate distribution of ZBP1 correlates with its ability to bind the Z-conformation, since single or double mutations of the Z-DNA contacting residues in ZαZBP1 (N46D and Y50A) resulted in a distribution similar to that of ZBP1ΔZα.
No such effect was observed with analogous mutations of ZβZBP1, even if the complete domain was deleted. It appears that ZαZBP1 exerts a dominant effect over ZβZBP1 with respect to the localization. This result could not be expected from the in vitro experiments, in which both Zα and Zβ appeared to bind Z-DNA with comparable efficiency. However, deletion of Zβ or mutation of its presumptive Z-DNA contacting residues N141 and Y145 resulted in accumulation of protein in the nucleus, indicating that a functional ZβZBP1 is involved in nuclear export of ZBP1. Importance of nuclear export is further supported by nuclear accumulation of full length ZBP1 after treatment with LMB, an inhibitor of nuclear export.
C-terminal ZBP1 deletion mutants (lacking exons 6–10), irrespective of the presence or absence of ZαZBP1
, showed a homogenous distribution within the cell, including the nucleus, similar to that of GFP. The C-terminal truncated ZBP1E1-5 and ZBP1E1-5ΔZα fused to GFP have a predicted molecular mass of 52 and 44 kDa, respectively. They appear to be too large to pass the nuclear pore by diffusion, where the cut-off is believed to be between 20 and 40 kDa (38
). This indicates that there might be another nuclear export signal apart from ZβZBP1
in the C-terminus of ZBP1. Western blot analysis showed that all ZBP1-EGFP fusion proteins were intact, demonstrating that the different observed fluorescence patterns were not a consequence of protein degradation.
The resemblance of the ZBP1-harboring granules with SGs and PBs led us to investigate whether ZBP1 might be actually a component of these. SGs and PBs are RNA granules which play important roles in mRNA turnover. Translationally silenced mRNA can be packed into these granules and either be degraded or stored and later exported. It is believed that SGs function as triage centers that sort, remodel and export mRNA either for subsequent decay (e.g. export to PBs) or for reinitiation of translation (25
It has been shown that SG formation can be promoted by the overexpression of SG proteins G3BP and TIA1 (27
). Co-transfection experiments showed that G3BP- and TIA1-marked granules are distinct from the ZBP1ΔZα marked granules. However, a close spatial association with overlapping contact areas between SGs and ZBP1ΔZα granules was often observed. A similar association was seen with ZBP1ΔZα granules and some PBs marked by the decapping enzyme DCP1a. Time lapse microscopy showed that these interactions are dynamic. Some ZBP1ΔZα granules were tethered to SGs or PBs over the complete observation period, while some only associated for a limited time. Upon contact with one another, SGs and ZBP1ΔZα granules often appeared to deform and reshape. They share some characteristic features, which include interaction with PBs, fusion and fission (25
The granules induced by the overexpression of SG protein G3BP are thought to be genuine SGs because they contain typical SG proteins such as PABP1, eIF4G, TIA1 and TIA1/R (27
). The finding that full length ZBP1 can actually localize into G3BP- and TIA1-marked SGs only after cells have been subjected to environmental stress, i.e. heat shock and arsenite exposure, indicates that G3BP- and TIA1-containing granules in unstressed transfected cells contain ‘stress-independent’ components that are assembled following the overexpression of these proteins. Other proteins such as ZBP1 enter these ‘pre-formed’ granules only after cells have been subjected to environmental stress (stress-dependent).
Inhibitors of translational elongation, such as emetine and cyclophosphamide, impede the disassembly of polysomes and dissolve SGs and PBs. Since mRNA shuttle between SGs and polysomes, this leads to a shift in the equilibrium of mRNAs towards the polysome fraction, resulting in the disintegration of SGs, because mRNA are integral components of SGs and PBs (24
). Interestingly, treatment of cells with emetine led to the disassembly of both SGs and ZBP1ΔZα granules, indicating that mRNA may also be an integral component of the latter. It will be interesting to determine other components of ZBP1ΔZα granules and analyze whether proteins and RNA, which are found in SGs and PBs, are also present in ZBP1ΔZα granules. Remarkably, exposure of cells to arsenite and heat shock dissolved the ZBP1ΔZα granules. The dynamic nature of ZBP1ΔZα granules indicates that these granules do not represent mere aggregates of misfolded proteins. A similar phenomenon has been observed for the AU-rich element binding protein tristetraprolin (TTP). TTP was recruited to SGs after FCCP-induced energy starvation but excluded form SGs in arsenite treated cells. Overexpression of TTP resulted in the formation of SGs, which disassembled after arsenite treatment (41
). The exclusion of TTP from SGs was shown to be regulated by the phosphorylation of 14-3-3 proteins via the p38-MAPK/MK2 pathway (41
Due to the predominant cytoplasmatic localization of ZBP1 it appears that cellular RNA might be a more likely target than cellular DNA. However, as potential Z-DNA forming sequences are found in the genomes of some viruses, e.g. simian virus 40, adenoviruses (6
), herpes simplex virus and molluscum contagiosum virus (S. Rothenburg, unpublished), it seems also possible that a major target for ZBP1 is viral Z-DNA or Z-RNA. Indeed ZBP1 is strongly induced after immunostimulation by interferons (18
) and its expression is tightly associated with inhibition of Hepatitis B virus replication in a mouse model (42
). ZBP1 was further reported to be strongly induced after serum starvation (43
). A recent report demonstrated a strong induction of ZBP1 mRNA in mouse embryonic fibroblasts by transfected double-stranded DNA that was dependent on the protein kinase TBK1, a key regulator of the interferon response (44
). This indicates that ZBP1 participates in antiviral responses triggered by viral DNA independent of other sensors for nucleic acid like Toll-like receptors and the helicase RIG-1 (44
Additionally or alternatively, the natural targets for ZBP1, ADAR1, PKZ and E3L Zα domains might be structured RNA that resemble the Z-conformation or slightly differ from it. The latter view is supported by the finding that ZαZBP1 is more important in determining the subcellular localization than ZβZBP1 despite comparable behavior of both domains in in vitro assays for the induction and binding of Z-DNA. This implies that ZαZBP1 might recognize the physiological target(s) better than ZβZBP1.
In this study we demonstrate that ZBP1 isoforms show different subcellular localizations, controlled by its first Z-DNA binding domain. While full length ZBP1 shows a cytosolic finely punctate distribution, the ZBP1ΔZα isoform as well as the mutants N46D and Y50A localize in large granules. ZBP1ΔZα granules are distinct from but interact dynamically with granules formed by SG and PB proteins. Furthermore, we identified full length ZBP1 as a previously unknown component of SGs induced by heat shock and arsenite treatment. The inducible redistribution of ZBP1 into SGs on one hand, and the association and dynamic interaction of ZBP1ΔZα with granules containing G3BP, TIA1 and DCP1a on the other hand, support the notion that ZBP1 isoforms play distinct roles in the regulation of mRNA metabolism and/or in the cellular defense system against viruses.