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HAX-1 was originally described as a factor interacting with hematopoietic cell-specific Src kinase substrate (HS-1). HS-1 is involved in B-cell receptor signaling, which includes its translocation to the nucleus upon phosphorylation . Subsequently, HAX-1 was shown to associate with a number of diverse proteins involved in several vital processes, such as apoptosis [2-5], cell migration [6, 7], regulation of calcium homeostasis [8, 9], interactions with viral proteins involved in cell survival [10-13] and ABC transporters . The number and scope of HAX-1 interactions suggests that its role in the cell must be significant, although probably complex . Its knockout in mice leads to postnatal lethality , while homozygous mutations in humans result in severe congenital neutropenia , a condition characterized by a paucity of neutrophils caused by maturation arrest in the bone marrow. Conversely, HAX-1 over-expression has been reported in several types of human cancer [7, 18, 19].
In addition to its other properties, which have hitherto attracted most attention, HAX-1 was also shown to interact with the 3′ UTRs of two transcripts: one encoding the cytoskeletal protein vimentin  and the second encoding an enzyme involved in base excision repair, DNA polymerase β (POLB) . In both cases, the interacting motif consisted of a stable hairpin, although there were no particular similarities in the structure. The transcript-binding properties of HAX-1 raise several questions regarding the role of these interactions in control of mRNA expression and the cellular compartment in which such binding takes place. HAX-1 has previously been described as localizing mainly in mitochondria [1, 4, 20], but also in the ER [9, 10], lamellipodia  and nucleus . One early report describes its localization in the nuclear membrane , but this observation has not been confirmed in later research. Data obtained by our group suggest that HAX-1 is instead associated with the nuclear matrix . Nuclear localization of HAX-1 was also demonstrated in systemic sclerosis fibroblasts  and breast cancer cells , which leads to the assumption that the observed accumulation may be disease-dependent.
The results presented here show that HAX-1 is in fact a nucleocytoplasmic shuttling protein. Nuclear localization was shown to vary depending on the isoform of the protein. The nuclear export of HAX-1 was shown to be dependent on exportin 1 (XPO1, a human homolog to yeast Crm1), and two nuclear export signals (NES) were identified in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after specific cellular stresses, mainly after sodium arsenite treatment. Intriguingly, upon arsenite stress, HAX-1 status affected POLB mRNA levels, while in normal conditions this effect was not observed. These results and the results of the HAX-1 tethered assay suggest that the putative role of HAX-1 in post-transcriptional regulation of expression may consist of affecting mRNA levels of the bound transcripts, possibly as a response to specific cellular stress.
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- Experimental procedures
- Supporting Information
Although HAX-1 protein is known to be involved in processes as crucial as apoptosis, cell migration and the regulation of calcium homeostasis, its mechanisms of function are largely unknown. Its interaction with the 3′ UTR of some mRNAs indicates a role in post-transcriptional regulation, but this function has been rather poorly studied to date. Here we report HAX-1 nuclear localization, its nucleocytoplasmic shuttling, co-localization with P-bodies and its impact on bound mRNA levels, which may help to elucidate its role in post-transcriptional regulation of expression.
In addition to many other cellular localizations, HAX-1 has been detected in the nucleus [15, 21]. In this study, we have established that the nuclear presence of HAX-1 is much more common than previously described and varies between isoforms; most of the human and rat isoforms localize to the cytoplasm, but some (human isoform IV and its rat counterpart isoform VII as well as rat isoform VI) are present in the nucleus. The importance and role of the primarily nuclear isoforms may be modest, as expression of isoforms other than isoform I is low , but, even for the cytoplasmic isoforms, nuclear localization may be detected in a portion of the analyzed cells. Furthermore, nuclear retention of human isoforms I, II and III was observed after LMB treatment, suggesting that HAX-1 is a nucleocytoplasmic factor and its nuclear export is mediated by XPO1. This assumption was confirmed by HAX-1–XPO1 co-immunoprecipitation.
Intriguingly, some GFP-fused HAX-1 isoforms localized in discrete foci in the nucleus, especially after LMB treatment. This observation suggests that HAX-1 may be present in specific nuclear bodies. Identification of these structures may help to unravel the specific nuclear role of HAX-1.
The findings presented may shed new light on the known inhibitory effect that HAX-1 exerts on the Rev protein of human immunodeficiency virus (HIV) . Rev facilitates XPO1 (Crm1)-dependent nuclear export of viral unspliced mRNAs containing the Rev response element. HAX-1 interacts with Rev and inhibits nuclear export of Rev response element RNAs . In the same study, HAX-1 was found to alter the intracellular localization of Rev from nuclear to cytoplasmic. These results were interpreted to indicate Rev trapping in the cytoplasm mediated by direct interaction with HAX-1. HAX-1 nucleocytoplasmic shuttling and its interaction with XPO1 suggests that the inhibitory effect of HAX-1 on Rev may involve more than a simple sequestration in the cytoplasm, and may result from interference with formation of the Rev–XPO1 exporting complex.
Quantitative analysis of the nuclear presence of HAX-1-GFP fusions displayed statistically important differences between isoforms before and after LMB treatment. The localization of isoform I was most affected by LMB, while localization of isoform IV remained unchanged. More subtle differences were detected for localization of isoforms II and III. These results indicate that the pool of shuttling HAX-1 consists mostly of isoform I.
Interestingly, HAX-1 nuclear translocation is never complete, and even for the most nuclear isoform IV and/or localization of the other isoforms after LMB treatment, there is always a vast proportion of the protein that remains in the cytoplasm.
In this study, two NES in the HAX-1 sequence were identified: a consensus NES in the C-terminal part of the protein, and a second non-consensus NES, containing a cluster of hydrophobic amino acids at positions 79-88. The double mutant in which both signals are mutated is predominantly nuclear.
Additionally, the N-terminal part of HAX-1 appears to have some influence on nuclear/cytoplasmic shuttling. The N-terminus contains a stretch of hybrophobic amino acids (3-13 amino acids) that resembles an NES and is absent from the frequently nuclear human isoform III and nuclear rat isoform VI.
There is no apparent nuclear localization signal within the HAX-1 sequence, which is not that surprising as the majority of the protein localizes to the cytoplasm. Two of the known HAX-1 protein partners, HS-1  and prohibitin , are cytoplasmic proteins that translocate to the nucleus upon lymphocyte receptor activation or in the presence of estrogen, respectively. This suggests that HAX-1 may enter the nucleus in complex with other proteins and in response to external (or internal) stimuli.
Accordingly, HAX-1 nuclear accumulation was observed in response to specific stress, particularly arsenite treatment and, to a lesser extent, heat shock, while H2O2, vinblastine, UV light and thapsigargin (an ER stress inducer) did not affect HAX-1 nuclear accumulation. It may be hypothesized that arsenite-induced oxidative stress inhibits XPO1 function , resulting in HAX-1 accumulation. However, strong oxidative stress induced by H2O2 treatment had no effect, indicating that arsenite causes more specific alterations in the cellular metabolism, to which HAX-1 responds.
HAX-1 nuclear accumulation after specific stress suggested its possible involvement in post-transcriptional regulation as part of the stress response. The POLB transcript is a known HAX-1 mRNA target. POLB expression was shown to be affected by arsenite treatment , and POLB enzyme was reported to participate in DNA repair after arsenite-induced damage . In this study, HAX-1 status was shown not to affect POLB mRNA levels under normal conditions, but a statistically significant increase in POLB mRNA levels was detected in the HAX-1-silenced cell line after arsenite stress. This result indicates that HAX-1 binding to the specific mRNA causes a decrease in the amount of this mRNA. As reported previously, the binding of HAX-1 to the instability-conferring element in the POLB mRNA  further supports these deductions. As HAX-1 binds to the 3′ UTR of the transcripts and lacks a DNA-binding domain, its effect on mRNA is most likely post-transcriptional. The effect of HAX-1 on POLB mRNA appears to be modest, but one should bear in mind that POLB expression is tightly regulated and any alteration may be detrimental. As POLB over-expression leads to chromosomal instability and an increased mutagenic rate [33, 34], the existence of mechanisms to combat this effect seems probable. Accordingly, HAX-1-mediated down-regulation may be an element of post-transcriptional fine-tuning of the stress response.
The results of HAX-1 tethering to the reporter mRNA corroborate the above conclusions. Again, any regulation observed in this reporter system is post-transcriptional. A decrease in the luciferase reporter was observed at the protein and mRNA levels, suggesting that HAX-1 affects mRNA stability. Intriguingly, HAX-1 was recently shown to interact with Pelota protein , a human homolog of yeast Dom34, which is involved in the ‘No-Go’ mRNA decay pathway. No-Go recognizes translation elongation stalling and targets the mRNA for endonucleolytic cleavage. A role of HAX-1 in this process remains to be established; it may be hypothesized that HAX-1 recruits Pelota to specific transcripts or mediates mRNA 5′ degradation after endonucleolytic cleavage, which may be linked to its co-localization with 5′-decay factors in P-bodies. However, at this point, no mechanism can be proposed to explain these findings, and further studies are required to clarify the role of HAX-1 in mRNA decay.
HAX-1 co-localization with the P-body marker further strengthens the possibility of its involvement in mRNA processing. Although their role remains controversial, P-bodies are thought to be involved in post-transcriptional activities such as decapping, degradation, miRNA silencing and storage of mRNA. Although no interaction with any P-body protein has been detected so far for HAX-1, its co-localization with these structures suggests involvement in mRNA processing, consistent with the data showing its influence on POLB and reporter mRNAs.
P-bodies are dynamically linked to and partially share protein content with stress granules , but, despite the co-localization with P-bodies, HAX-1 was found not to be present in stress granules. This result indicates that it has no specific role in storage and protection of translationally active mRNAs during stress.
The results presented above raise several questions concerning HAX-1 biology and its functions other than functions directly related to apoptosis, cell migration or calcium homeostasis. The functional significance of the nuclear presence of HAX-1 and its nucleocytoplasmic shuttling remains to be established. The HAX-1 transcript-binding properties suggest that its nuclear role may be associated with processing of specific mRNAs. The results obtained here suggest that this mRNA processing may consist of regulation of the stability of specific transcripts, possibly as an element of fine-tuning of the cellular stress response. This conclusion is supported by the presence of HAX-1 in P-bodies. There are several examples of nucleocytoplasmic shuttling proteins that are present in P-bodies and that play a role in post-transcriptional regulation: HuR , rck/p54 (DDX6) , DDX3 , the Rpb4p subunit of RNA polymerase II , CPEB  and Pat1b . HAX-1 may represent another example, and further studies are required to clarify its role in mRNA processing.