The results presented here provide evidence that evolutionarily conserved sequences within the 3′UTR are good candidates for functional regulatory elements. Indeed, most of the HCRs studied here had a significant effect on gene expression at the posttranscriptional level. The approach we employed is not intended to be exhaustive; some important regulatory elements will be missed, since they are species specific and not conserved during evolution. However, this phylogenetic approach serves as a means of focusing on those sequences that are likely to have an important regulatory function. Clearly, certain nonconserved regions will also have posttranscriptional effects on gene expression, but by focusing on sequences that have not diverged, the search for such regulatory elements becomes less random. For example, of the 10 HCRs studied, each altered gene expression at the posttranscriptional level. Three were well characterized, and we were able to show that the HCRs had all of the functions previously ascribed to the 3′UTRs (c-fos, c-myc, and transferrin receptor), localizing the functional regions to those that had not diverged significantly from chicken to mouse. Conservation did not provide insight into the type of regulatory function, since the 10 HCRs had different effects. Indeed, only a subset of possible functions were tested here and other conditions may show as-yet-unidentified HCR effects. Our findings demonstrate that HCRs are potential regulatory regions that provide an important check-and-balance on transcription, both under steady-state conditions and especially in response to environmental stresses such as changes in mitogen levels or oxygen deprivation.
Three HCRs were chosen to validate our method of analysis which we then extended to the study of seven relatively uncharacterized HCRs. These HCRs were selected for study because of their evolutionary conservation and because the protein products encoded by the mRNAs with which they are associated have known functions in growth control, differentiation, and cancer. For all three well-characterized HCRs (c-fos, c-myc, and TfR), the reported effects of the 3′UTR on mRNA destabilization were demonstrated. The system was then used to compare and rank the effects of the diverse HCRs on posttranscriptional regulation. This was possible because all of the other vector components, such as the CMV minimal promoter, 5′UTR, GUS reporter, and polyadenylation signal, remained constant.
Five of the ten HCRs tested led to a two- to tenfold reduction in mRNA steady-state levels. With the notable exception of the 3′UTR of α-globin, which increases mRNA stability (85
), 3′UTR regulatory sequences have generally been proposed to reduce mRNA stability, which is in agreement with our findings (68
). Thus, HCRs may provide a safeguard against overexpression of proteins with a role in cell growth control and differentiation, thus maintaining a critical balance of such proteins.
The observed reduction in certain mRNA steady-state levels could be due in part to specific sequence motifs, such as the well-defined AU-rich elements (AUREs) (8
). Of interest is the finding that HCRs located in 3′UTRs are generally AU-rich (particularly U-rich) relative to the non-HCR region of the 3′UTRs (20
). These sequences are known to be present in several unstable mammalian oncogene and lymphokine mRNAs (7
), as well as in some of the HCRs tested here (c-fos, c-myc, and bcl2). However, since the degree of instability conferred by those HCRs that harbor AUREs differs, context is likely to play a major role in their function. In fact, all five of the HCRs studied here that contain AUREs extend beyond those sequences and include additional sequences that are highly conserved across species. Such domains could well include other destabilizing motifs or constitute binding sites for modulators of the AURE-specific degradation machinery. Indeed, the c-fos HCR, which exhibited the greatest degree of destabilization by far, is more than threefold the size of the three consecutive AU-rich domains encompassing the AURE; in addition it contains a 20-nucleotide U-rich sequence that has been shown previously to play a role in the deadenylation of the message leading to degradation (86
). Regulation of polyadenylation is thought to play an essential role in mammalian mRNA decay as in yeast cells (16
) and can be initiated by shortening the poly(A) tail, followed by decapping and 5′- and 3′-exonucleolytic degradation of the transcript (79
). Although it is clear that the number, spacing, and conserved sequences flanking AUREs can all affect their destabilization potential, the rules that govern these effects and the roles of independent but synergistic domains remain unknown. The context within a given mRNA is important not only to AURE function but also to cell physiology. For example, when primary T cells are activated by exposure to antibodies to the CD3 and CD28 receptors, several AU-rich mRNAs, including granulocyte-macrophage colony-stimulating factor, interferon, and interleukin-2, are stabilized, whereas the AU-rich c-myc mRNA is not (50
). Although the c-fos, c-myc, and transferrin 3′UTR sequences are all well-known to alter mRNA stability, the remaining 3′UTR sequences could alter mRNA steady-state levels by other mechanisms. The Hermes HRSpuro-GUS retroviral vector described here will allow the rapid analysis of mutant and truncated HCRs and HCRs located 3′ and 5′ to the reporter. It should prove useful in comparing diverse HCRs under a range of conditions and in the elucidation of the properties critical to AURE function in mammalian cells.
A second well-documented mechanism that leads to mRNA decay is endonucleolytic cleavage within the transcript. Examples include the destabilization of mRNAs encoding TfR and insulin-like growth factor II (55
), both of which contain stem- loop structures in the 3′UTR. In the case of TfR, the HCR contains five stem-loop structures (A to E) designated as iron-responsive elements (IREs). These structures bind transacting proteins (iron regulatory proteins) that mask the endonucleolytic cleavage site present between IREs C and D, thereby stabilizing the mRNA (3
). Whether the presumed destabilization induced by the remaining HCRs studied here is determined by sequences that decrease the binding of poly(A) binding protein, decrease poly(A) length, or provide sites for endonucleolytic cleavage remains to be determined, and the approach described here will enable such an analysis.
The amount of protein produced by translation of an mRNA is regulated at multiple levels (24
). The mRNA contains regulatory elements that interact with transacting factors that modulate translation initiation, elongation, and termination. The rate of initiation is known to be strongly influenced by certain sequences (61
) or secondary structures in the 5′UTR of mRNAs, as in the case of ODC (31
) or ferritin (30
). In contrast to 5′UTR-mediated control, the mechanisms by which 3′UTRs influence the translation process are understood at a more rudimentary level. Recent studies in yeast cells suggest that 3′UTRs may alter translation by a looping of the mRNA via the binding of the poly(A) tail and its binding protein (PABP) to the initiation factor eIF4G, which is part of the cap-binding protein complex eIF4F (77
). Biochemical evidence suggests that a similar interaction may occur in mammals (46
). Such an approximation of the terminal portion of the 3′UTR with the 5′ end of the mRNA could explain both the negative and positive effects of 3′UTRs on translation efficiency. Alternatively, the 3′UTR could be bound by proteins that lead to the sequestration of the mRNA into an untranslatable mRNP particle (73
). The best-documented cases for translational control via 3′UTRs are the gradients of regulatory molecules that lead to pattern formation in developing Drosophila
) and the temporal control of expression of erythroid 15-lipoxygenase mRNA in mammals (60
Only two of the 3′UTR sequences tested here had marked effects on translation efficiency. The c-fos HCR repressed translation fivefold. The effect of the c-fos 3′UTR on translation repression had been previously noted in nondividing Xenopus
) but not in mammalian proliferative somatic cells. Most 3′UTR sequences, like the tra-2 and GLI element (TGE) within the GLI 3′UTR (40
), repress translation. The vimentin HCR sequence enhanced translation twofold. With the possible exception of the amyloid protein precursor mRNA (17
), reports of 3′UTRs that promote translation are rare. To test whether the findings (90
) of a Y-shaped secondary structure localized within the “vim a” domain (Fig. ) might mediate the effects of the vimentin HCR on translation, we divided the HCR in half and introduced each half separately into different cell populations. The stimulation of translation of each half was reduced to control (−)HCR levels, indicating that the structure described by Zehner and colleagues does not confer the observed translational induction. Of note is the finding that the HCR of vimentin has a dual effect. This HCR not only stimulates translation twofold but also decreases mRNA levels to 70% of the control levels. The relative roles of these two opposing HCR-mediated mechanisms of controlling mRNA stability and translation could change in response to environmental cues and play a critical adaptive role, in agreement with proposed models that invoke coupling of mRNA stability to translation (80
Cells are known to regulate the expression of different genes in response to changing environmental conditions, such as nutrient or oxygen supply. Our data indicate that HCRs act as sensors and can alter gene expression in response to such stresses at a posttranscriptional level. Four HCRs (Ran, ODC, fibronectin, and HuD) responded to an increase in mitogens by increasing protein levels 1.5- to 2-fold. Here we show that the ODC HCR can alter gene expression independently of 5′ or coding components of the endogenous mRNA. Therefore, the translational stimulatory effect of the HCR is evident even in the absence of the endogenous 5′ UTR of ODC, which is known to contain an extensive secondary structure that represses the translation of ODC, an effect partially relieved by its 3′UTR (31
). Although increases in proteins in response to serum stimulation have also been previously reported for Ran (11
) and fibronectin (62
), evidence that these increases could be controlled at a posttranscriptional level as shown here has not been apparent. HuD has not previously been shown to be regulated at a posttranscriptional level. Whether regulation occurs at the level of mRNA stabilization or translation remains to be determined. Nonetheless, these findings demonstrate that HCRs can be sensors of mitogen concentrations, leading to altered protein levels that may be essential to cell survival.
Hypoxia and reoxygenation often accompany injury, ischemia, and stroke. In addition, evidence is also accumulating that tumor hypoxia plays an integral role in the malignant progression of cancers (29
). Solid tumors typically have regions that are necrotic, and this can be accompanied by perinecrotic hypoxia. The expression of many genes is altered at the transcriptional level in response to hypoxia, and this regulation is mediated in part by the heterodimeric transcription factor, hypoxia-inducible factor 1 (HIF-1) (28
). Other transcription factors have been implicated in the hypoxia response, such as c-fos, which together with c-jun presumably acts in the AP-1 transcription complex, which has been shown to be partially responsible for the expression of tumor metalloproteases stromolysin or type 1 collagenase (12
). We show here that the induction of c-fos by hypoxia is regulated not only at the transcriptional level (84
) but also at the posttranscriptional level by the HCR in the 3′UTR. This mode of c-fos regulation has not been previously reported. It will be of interest to determine whether the underlying posttranscriptional regulatory mechanisms are similar to the ones described for the hypoxic induction of VEGF (49
) or erythropoetin (54
). The importance of this class of posttranscriptional regulation of genes in response to hypoxia has become apparent because the loss of the tumor suppressor VHL results in the loss of this type of regulation (25
). Given the magnitude of the effect we observe, additional hypoxia-responsive HCRs could be identified by functional genomic screening of retroviral cDNA libraries and FACS analysis (34
). Such HCRs may provide a molecular switch that responds to the inhibitory conditions in the microenvironment of solid tumors.
Finally, the modulation of levels of 3′UTR expression through the use of regulator retroviruses such as RetroTet RTAb(+) and RetroTet RTRb(−) will now facilitate the in-depth analysis of 3′UTR sequences with a role in growth inhibition and differentiation, such as those previously described (15
), since expression can be suppressed during cell expansion and induced specifically at the time of analysis. Moreover, our inducible retroviral system should allow the study of mRNA decay kinetics without perturbing cellular physiology, as is the case for transcription inhibitors such as actinomycin D or inducible systems based on transient expression of the c-fos promoter after serum stimulation. The inducible expression of HCRs will facilitate control of the concentration of HCR-containing mRNA molecules in the cell by varying the amount of Tet in the culture medium (44
). An excess of exogeneous HCR molecules could titrate out UTR regulatory binding proteins and modify the steady-state level of expression of the endogenous gene as reported for creatine kinase B (9
) and ODC (51
), which could in turn lead to a pleiotropic effect on gene expression and the consequent alteration of cell physiology. The use of the reporter retroviral vector used here should diminish the risks of overexpressing the HCR-containing reporter compared to transient-transfection experiments, due to the low copy number of transgenes introduced and the use of a minimal promoter. Moreover, superinfection with the Tet-regulatable transrepressor retrovirus can be used to further decrease transcription and thus HCR-mRNA dosage.
The findings described here may have applications to the treatment of viral and malignant diseases. Tet-inducible overexpression of exogenous HCR sequences could provide a means to alter the balance of genes involved in growth control or hypoxia. Posttranscriptionally mediated therapies could be designed that mimic mechanisms used by viruses. For example, competition between the c-fos 3′UTR instability elements and the papillomavirus late mRNAs for the same poly(U) binding proteins has been postulated to lead to elevated Fos protein levels in infected cells (71
). Thus, HCR expression in a time- and dose-dependent manner could be useful as an adjunct to traditional antiviral and cytostatic agents.