The rapid, dramatic increases in flAlu RNA in response to viral infection, cell stress, and translational inhibition have raised the possibility that these transcripts serve a physiological role (19
). Since each of these treatments also affects protein synthesis, we tested the effects of flAlu RNA on translation by using the expression of a reporter gene. Overexpression of flAlu RNA significantly increased the expression of a luciferase reporter. Negative controls, particularly one employing scAlu RNA, indicated that the stimulated expression of this reporter was not an artifact of RNA overexpression but was attributable to the specific activity of flAlu RNA. Furthermore, the levels of Alu overexpression required to cause an increase in luciferase were comparable to the levels of flAlu RNA induced by cell stress and viral infection, suggesting that endogenous levels of flAlu RNA have similar effects on translational expression. Similarly, levels of flAlu RNA overexpression comparable to those induced by cell stress and viral infection also caused a decrease in PKR activity, suggesting a mechanism for the effects of flAlu RNA on translational expression.
Viral infection and cell stress alter translation by multiple and redundant pathways, including pathways which initially increase and subsequently decrease eIF-2α phosphorylation, thereby first repressing and then derepressing translational initiation (12
). Significantly, in both cases, dephosphorylation of eIF-2α to reactivate translational initiation parallels the appearance of entirely new mRNA cohorts encoding viral or heat shock proteins. Inhibition of translational elongation has previously been reported to decrease eIF2α phosphorylation (42
). In agreement with that result, we observed that cycloheximide treatment caused a very rapid decrease in PKR activity, which may lead to a decrease in eIF-2α phosphorylation. Presumably, an increase in translational initiation would be an initial, regulated cellular response to a decreased rate of translational elongation. In any event, PKR activity was altered by each of these three cellular treatments, which also increase the level of flAlu RNA.
We further observed that flAlu RNA bound PKR both in vitro and in vivo. In gel mobility shift assays, Alu RNA formed discrete complexes with PKR. The relationship of these complexes to either the binding of PKR to Alu RNA in vivo or the inhibition of PKR by Alu RNA is uncertain. However, under competitive binding conditions, preformed flAlu RNA complexes were relatively stable compared to those formed by VAI RNA, a well-studied PKR antagonist. Presumably, any RNA with a sufficient secondary structure could bind to PKR in vitro (7
), but many such RNAs, in the form of stable ribonucleoprotein structures, would not be available for PKR binding in vivo. In agreement with this suggestion, both VAI RNA and flAlu RNA bound PKR in vivo, but we did not observe any interaction between PKR and 7SL RNA, which is far more abundant than is flAlu RNA. As a component of fully formed, functional signal recognition particles (SRPs), 7SL RNA would be protected from PKR binding. The most active PKR inhibitors, as exemplified by VAI RNA, may be highly structured RNAs which do not serve functions that require being tightly sequestered in ribonucleoprotein structures. flAlu RNA is known to bind only the two smallest SRP proteins, presumably making it accessible to PKR in vivo.
flAlu RNA was equally as effective as was VAI RNA in antagonizing the activation of PKR in vitro; more importantly, overexpressed flAlu RNA also antagonized virus-induced activation of PKR in vivo. Interestingly, overexpressed flAlu RNA antagonized PKR activation to almost the same degree as did far higher concentrations of VAI RNA, leading us to conclude that flAlu RNA is indeed a potent PKR inhibitor. Higher levels of overexpressed scAlu RNA did not cause this inhibition, showing that the antagonism of PKR by flAlu RNA is not an artifact of gross overexpression but depends upon the structure of flAlu RNA.
These observations suggest that endogenous flAlu RNA affects translational expression by inhibiting PKR activation. We have not yet tested this possibility directly. However, cell stress, cycloheximide treatment, and viral infection changed PKR activity, which presumably modulates changes in protein synthesis caused by these same treatments. We also observed a rather simple dose-response relationship between the abundance of exogenously expressed flAlu RNA and the levels of both transiently coexpressed luciferase activity and PKR activity. Furthermore, the increased levels of flAlu RNA caused by viral infection, translational inhibition, and cell stress were similar to the levels of flAlu RNA overexpression that stimulated expression of the luciferase reporter and concomitantly decreased PKR activity. Thus, we consider it a possibility that increases in flAlu RNA caused by these three cellular treatments affect PKR activity and consequently protein synthesis.
As a host defense against viral infection, PKR is activated by dsRNA to phosphorylate eIF-2α, thereby blocking protein synthesis (8
). PKR is also an important signal transducer for interferon and cytokine induction of gene expression through regulation of transcription factors NF-κB and IRF-1 (25
). Viral counterstrategies to block PKR activation include the synthesis of massive quantities of small RNAs, such as VAI RNA in the case of adenovirus, to antagonize PKR’s activation (8
). In the most thoroughly studied case of adenovirus, viral gene products direct the increase in Alu transcription after infection (36
). By binding PKR and antagonizing its activation, these induced flAlu transcripts may provide another viral defense against PKR activation by the host. There are already many known viral strategies to counter host defenses, and the existence of yet another is not surprising (41
). Of course, VAI RNA encoded by adenovirus accumulates to a much higher level than does short-lived flAlu RNA and would therefore be expected to serve as a far more effective PKR antagonist. However, virus-encoded pathways are often deleterious to the host cell. A cellular PKR antagonist would not need to achieve the level of PKR antagonism provided by VAI RNA. It is noteworthy that VAI deletion mutants, although impaired, remain viable, indicating that there are redundant pathways to overcome host defenses (41
). Although the physiological function of flAlu RNA cannot be to enhance viral infectivity, viruses typically co-opt normal cellular regulatory mechanisms.
As discussed above, a decrease in PKR activity is plausibly an initial cellular response to the inhibition of translational elongation caused by drugs such as cycloheximide. More than 20 years ago, Reichman and Penman identified a factor, termed an activator, which stimulates translational initiation (37
). They demonstrated that this activator is an RNA with a half-life of about 1 h, which is approximately the short lifetime of flAlu RNA (6
). Like flAlu RNA, activator RNA is induced by both heat shock and cycloheximide treatment of cells. Comparing the results of Goldstein et al. (16
) to those of Liu et al. (28
), the transient increases in both activator RNA and flAlu RNA levels in response to cycloheximide are virtually identical. We suspect that flAlu RNA is this translational activator. Retrospectively, the antagonism of PKR activation by the increased levels of flAlu RNA caused by cycloheximide treatment provides a mechanistic explanation for the observed biochemical activity of activator RNA upon translational initiation.
Hemin-regulated initiation factor-2 kinase, a PKR homolog, phosphorylates eIF-2α in response to heat shock, but PKR itself has no known role in the heat shock response (10
). During long-term heat shock, PKR changes from a soluble form to an insoluble form (11
). We observed transient increases in PKR activity during heat shock recovery. However, the kinetic relationship of these changes in PKR activity to transient increases in flAlu RNA abundance during heat shock recovery is not evident. At present, any proposed function that PKR may serve during the heat shock response would be entirely speculative; therefore, identifying the effects that flAlu RNA may cause by acting on PKR during this period is even more problematic. However, complex changes in translational expression occur during both heat shock and heat shock recovery and, in part, these changes are regulated by changes in eIF-2α phosphorylation (12
). Consequently, PKR and its regulators are almost certainly involved in the heat shock response. As suggested above, activator RNA may be a PKR regulator which is induced by heat shock (6
Ideally, the possibility that Alu RNA fulfills a physiological role would be tested by genetics. Because of their extraordinary copy number, Alu RNAs are not amenable to classical genetic tests. In Tetrahymena spp., a small, Pol III-transcribed RNA rapidly accumulates after heat shock and is required for the establishment of thermal tolerance (13
). Interestingly, this transcript, like human Alu RNAs, is related to 7SL RNA. We do not know whether there is any relationship between the function of this gene and the present observations concerning flAlu RNA. However, there are several intriguing parallels between these two systems and certainly the Tetrahymena results provide strong precedence for the functionality of small heat shock-induced RNAs.
This proposed role for human Alu RNA potentially explains some unusual evolutionary features of mammalian SINEs. (i) There is no homology between human Alu RNAs and many other mammalian SINEs, such as rabbit SINEs (39
). Either SINEs are functionless or their function(s) does not depend upon sequence but upon some other, higher-order structure. However, entirely nonhomologous mammalian SINE RNAs could antagonize PKR since the binding of PKR to duplex RNA regions does not depend strongly on the sequence but rather requires a minimum number of base pairs of sufficient base pair fidelity (8
). Interestingly, the dimeric structure of human Alu elements may make their transcripts unusually potent PKR antagonists since each flAlu RNA potentially binds two molecules of PKR. The results from gel shift assays support this intriguing possibility. (ii) Individual mammalian SINEs are weakly promoted; therefore, their expression is easily repressed (6
). Nevertheless, the extremely large number of SINEs guarantees that many elements are always in active chromatin domains, thereby permitting a robust transcriptional response in all cell types despite the weakness of their individual promoters (6
). (iii) The short lifetime of SINE transcripts, which lack other normal cellular functions, makes them ideally suited to signal PKR. The abundance of a long-lived RNA serving an essential constitutive function could not be rapidly and significantly increased without disrupting that function.
Although an exact physiological role for Alu RNA and more generally mammalian SINE RNAs remains to be determined, overexpressed Alu transcripts stimulate translation and almost certainly do so by antagonizing PKR activation. Adaptation of these effects to regulate translational expression could provide a significant selective advantage for the maintenance of SINEs within the mammalian genome. The induction of Alu RNAs by cell stress and other treatments and the association between Alu RNA and PKR suggest that this potential selective advantage is being exploited.