Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.     

Tethering KSRP, a decay-promoting AU-rich element-binding protein, to mRNAs elicits mRNA decay

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.     

Improved primate foamy virus vectors and packaging constructs

Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.     

A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus

The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation.     

Functional mapping of destabilizing elements in the protein-coding region of the Drosophila fushi tarazu mRNA

The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3' untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3' UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3' UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5'-elements depended on their position within the message.     

Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5'' noncoding region.

Poliovirus polysomal RNA is naturally uncapped, and as such, its translation must bypass any 5' cap-dependent ribosome recognition event. To elucidate the manner by which poliovirus mRNA is translated, we have determined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. We found striking differences in translatability among the altered mRNAs when assayed in mock-infected and poliovirus-infected HeLa cell extracts. The results identify a functional cis-acting element within the 5' noncoding region of the poliovirus mRNA which enables it to translate in a cap-independent fashion. The major determinant of this element maps between nucleotides 320 and 631 of the 5' end of the poliovirus mRNA. We also show that this region (320 to 631), when fused to a heterologous mRNA, can function in cis to render the mRNA cap independent in translation.     

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.     

Requirements for enhanced transgene expression by untranslated sequences from the human cytomegalovirus immediate-early gene.

BACKGROUND: The cytomegalovirus immediate early (CMV IE) promoter has been widely used for heterologous expression. Further enhancements of gene expression from this potent promoter may allow for the development of improved gene transfer strategies. We aimed to determine whether inclusion of the first exon (5' untranslated) and first intron of the CMV IE gene would increase heterologous transgene expression in primary target cells and to determine the sequences required for any observed increases. MATERIALS AND METHODS: Comparisons of reporter gene expression were made following transient transfection of vascular smooth muscle cells (VSMCs) with plasmids containing the first exon and intron from the CMV IE gene or deletional mutations. Comparisons were also made using a heterologous promoter (RSV). RESULTS: Gene expression from the CMV IE promoter was increased 5.7-fold in VSMC with the inclusion of the first exon and intron. Similar increases were seen with other target cells and from the heterologous RSV promoter. This increase was associated with an increase in steady-state mRNA. Deletion analyses demonstrated that the enhancement was dependent on the presence of the 5' portion of the first exon while deletion of large segments within the intron was associated with similar levels of expression compared with the parental plasmid. CONCLUSIONS: Inclusion of the first exon and intron from the CMV IE gene increases expression from the CMV IE promoter. This enhancement is seen with the heterologous RSV promoter and is associated with an increase in steady-state mRNA. Deletion analyses suggest that this enhancement is associated with inclusion of sequences within the 5' portion of the first exon and inclusion of an intron.     

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.     

Negative regulation of expression of the Bacillus megaterium bmlP1 gene by the bmlP1 3' flanking region

We report that the expression of the Bacillus megaterium bmlP1 gene is subject to negative regulation by the bmlP1 3' flanking region. This repression occurred both in B. megaterium and in Escherichia coli. When the bmlP1 promoter was replaced with a heterologous promoter or when the orientation of the bmlP1 3' flanking region was reversed, the inhibitory effect was still observed. However, the bmlP1 3' flanking region was unable to exert repression on a heterologous gene when fused downstream in either orientation, and it was incapable of acting in trans. Dot blot and Northern blot analyses revealed that the repression occurred at the RNA level. Deletion analysis showed that the regulatory site responsible for the repression is located within a 116-bp region immediately following the bmlP1 gene. Possible mechanisms for this repression are discussed.     

The human immunodeficiency virus type 1 Rev protein and the Rev-responsive element counteract the effect of an inhibitory 5' splice site in a 3' untranslated region.

A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5' splice site in a 3'UTR were shown to act synergistically, and this effect could also be moderated by the interaction of Rev and the RRE. These studies provide additional evidence that at least one mechanism of Rev action is through interactions with the splicing machinery. We have previously shown that base pairing between the U1 small nuclear RNA and a 3'UTR 5' splice site is required for inhibition of gene expression. However, experiments by J. Kjems and P. A. Sharp (J. Virol. 67:4769-4776, 1993) have suggested that Rev acts on spliceosome assembly at a stage after binding of the U1 small nuclear ribonucleoprotein to the 5' splice site. This finding suggests that binding of additional small nuclear ribonucleoproteins, as well as other splicing factors, may be necessary for the inhibitory action of a 3'UTR 5' splice site. These data also suggest that expression of the papillomavirus late genes in terminally differentiated keratinocytes can be regulated by a viral or cellular Rev-like activity.