HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Inhibition of translation in L-cell lysates by free polyadenylic acid: differences in sensitivity among different mRNAs and possible involvement of an initiation factor

Free polyadenylic acid specifically inhibits in vitro translation of naturally polyadenylated mRNAs in L-cell lysates. The polynucleotide affects the initiation of protein synthesis but has no apparent effect on elongation of polypeptide chains. Reovirus mRNA, naturally devoid of a poly(A) tail, is much less sensitive to this inhibition than are naturally polyadenylated mRNAs. Reovirus mRNA that was polyadenylated in vitro is not more sensitive than normal reovirus mRNA. The degree of inhibition of translation varies for the different reovirus mRNA species. The addition of proteins contained in a high salt wash of ribosomes can mitigate the inhibition of translation of naturally polyadenylated mRNAs by free polyadenylic acid. Altogether these results suggest that the inhibition by polyadenylic acid may be mediated by its interaction with a cellular (initiation) factor. The various sensitivities exhibited by different mRNAs may indicate differences in requirement for this factor.     

Polyadenylation state of abundant mRNAs during Drosophila development

We have used a two-dimensional gel analysis of cell-free translation products to determine whether individual mRNAs present in Drosophila melanogaster embryos, larvae, pupae, and adults are predominantly polyadenylated or nonadenylated. While the majority of the embryonic mRNAs we detected exist mainly in the polyadenylated form, these mRNAs become more evenly distributed between the poly(A)+ and poly(A)- RNA fractions during postembryonic development. Although DNA:RNA hybridization experiments have indicated that Drosophila RNA populations contain a large group of rare class mRNAs restricted to the poly(A)- RNA compartment, this is not true for the 150 more abundant mRNA species analyzed by our methods. The histone mRNAs are the only abundant mRNA species which appear to be exclusively in the poly(A)- RNA class.     

Poly(A) tail shortening is the translation-dependent step in c-myc mRNA degradation.

The highly unstable c-myc mRNA has been shown to be stabilized in cells treated with protein synthesis inhibitors. We have studied this phenomenon in an effort to gain more insight into the degradation pathway of this mRNA. Our results indicate that the stabilization of c-myc mRNA in the absence of translation can be fully explained by the inhibition of translation-dependent poly(A) tail shortening. This view is based on the following observations. First, the normally rapid shortening of the c-myc poly(A) tail was slowed down by a translation block. Second, c-myc messengers which carry a short poly(A) tail, as a result of prolonged actinomycin D or 3'-deoxyadenosine treatment, were not stabilized by the inhibition of translation. We propose that c-myc mRNA degradation proceeds in at least two steps. The first step is the shortening of long poly(A) tails. This step requires ongoing translation and thus is responsible for the delay in mRNA degradation observed in the presence of protein synthesis inhibitors. The second step involves rapid degradation of the body of the mRNA, possibly preceded by the removal of the short remainder of the poly(A) tail. This last step is independent of translation.     

Auxin-regulated gene expression

During the 1960s a wide range of studies provided an information base that led to the suggestion that auxin-regulated cell processes--especially cell elongation--may be mediated by auxin-regulated gene expression. Indirect evidence from our work, based on the influence of inhibitors of RNA synthesis (e.g. actinomycin D) and of protein synthesis (e.g. cycloheximide) on auxin-induced cell elongation, coupled with correlations of the influence of auxin on RNA synthesis and cell elongation, provided the basis for this suggestion. With the availability of techniques for DNA-DNA and DNA-RNA hybridization, mRNA isolation-translation, in vitro 2D gel analysis of the translation products, and ultimately the cloning by recombinant DNA technologies of genomic DNA and copy DNAs (cDNAs) made to poly(A)+ mRNAs, we and others have provided direct evidence for the influence of auxin on the expression of a few genes (i.e. poly(A)+ RNA levels). Our laboratory has provided evidence for auxin's both down-regulating and up-regulating the level of a few poly(A)+ mRNAs out of a population of about 4 X 10(4) sequences that are not significantly affected by auxin. In our studies on auxin-regulated cell elongation, two cDNA clones (pJCW1 and pJCW2) were isolated which corresponded to poly(A)+ mRNAs that responded during growth transitions in a way consistent with a potential role of their protein products in cell elongation. These mRNAs are most abundant in the elongating zone of the soybean hypocotyl. Upon excision and incubation in the absence of auxin, these mRNAs deplete in concert with a decreasing rate of cell elongation. Addition of auxin to the medium results in both increased levels of these mRNAs and enhanced rates of cell elongation. These mRNAs do not deplete if auxin is added to the medium at the onset of excised incubation, and cell elongation rates remain high. We have isolated and sequenced genomic clones that are homologous to these cDNAs. Of the two genes sequenced, both genes are members of small multigene families. There are regions of high amino acid homology even though the nucleotide sequences are sufficiently different in these regions for cross-hybridization of the clones not to be observed. More recently others, especially Guilfoyle's laboratory, have shown that auxin selectively and rapidly influences the level of certain mRNAs and proteins. We have worked on other gene systems such as ribosomal proteins and possible cell wall proteins that are responsive to auxin; again the nature of regulation of expression of these genes is not known.(ABSTRACT TRUNCATED AT 400 WORDS)     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Autoregulation of poly(A)-binding protein synthesis in vitro.

The poly(A)-binding protein (PABP), in a complex with the 3'poly(A) tail of eukaryotic mRNAs, plays important roles in the control of translation and message stability. All known examples of PABP mRNAs contain an extensive A-rich sequence in their 5' untranslated regions. Studies in mammalian cells undergoing growth stimulation or terminal differentiation indicate that PABP expression is regulated at the translational level. Here we examine the hypothesis that synthesis of the PABP is autogenously controlled. We show that the endogenous inactive PABP mRNA in rabbit reticulocytes can be specifically stimulated by addition of low concentrations of poly(A) and that this stimulation is also observed with in vitro transcribed human PABP mRNA. By deleting the A-rich region from the leader of human PABP mRNA and adding it upstream of the initiator AUG in a reporter mRNA we show that the adenylate tract is sufficient and necessary for mRNA repression and poly(A)-mediated activation in the reticulocyte cell-free system. UV cross-linking experiments demonstrate that the leader adenylate tract binds PABP. Furthermore, addition of recombinant GST-PABP to the cell-free system represses translation of mRNAs containing the A-rich sequence in their 5'UTR, but has no effect on control mRNA. We thus conclude that in vitro PABP binding to the A-rich sequence in the 5' UTR of PABP mRNA represses its own synthesis.     

How strong is the case for regulation of the initiation step of translation by elements at the 3' end of eukaryotic mRNAs?

The belief that initiation of translation requires communication between the 5' and 3' ends of the mRNA guides--or misguides--the interpretation of many experiments. The closed-loop model for initiation creates the expectation that sequences at the 3' end of eukaryotic mRNAs should regulate translation. This review looks closely at the evidence in three prominent cases where such regulation is claimed. The mRNAs in question encode 15-lipoxygenase, ceruloplasmin, and histones. Vertebrate histone mRNAs lack a poly(A) tail, instead of which a 3' stem-loop structure is said to promote translation by binding a protein which purportedly binds initiation factors. The proffered evidence for this hypothesis has many flaws. Temporal control of 15-lipoxygenase production in reticulocytes is often cited as another well-documented example of translational regulation via the 3' untranslated region, but inspection of the evidence reveals significant gaps and contradictions. Solid evidence is lacking also for the idea that a ribosomal protein binds to and shuts off translation of ceruloplasmin mRNA. Some viral RNAs that lack a poly(A) tail have alternative 3' structures which are said to promote translation via circularization of the mRNA, but in no case has this been shown convincingly. Interpretation of many experiments is compromised by possible effects of the 3' structures on mRNA stability rather than translation. The functional-half-life assay, which is often employed to rule out effects on mRNA stability, might not be adequate to settle the question. Other issues, such as the possibility of artifacts caused by overexpression of RNA-binding proteins, can complicate studies of translational regulation. There is no doubt that elements at the 3' end of eukaryotic mRNAs can regulate gene expression in a variety of ways. It has not been shown unequivocally that one of these ways involves direct participation of the 3' untranslated region in the initiation step of translation.     

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.     

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.