Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1

The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.     

Sequence and translation initiation properties of the xenopus TGFbeta5, PDGF-A, and PDGF-alpha receptor 5' untranslated regions

The properties of the architecturally complex Xenopus laevis TGFbeta5, PDGF-A and PDGF-alpha receptor 5'UTRs were investigated. 5' extended cDNAs were obtained by 5'RACE, resulting in long 5'UTRs (478-710 nt) with multiple upstream AUGs (3-13), andthe potential to fold into stable structures. Injection studies suggested that the cloned PDGF-alphaR 5'UTR contains an intron. Splicing at potential 5' and 3' splice sites would result in a non-complex 5'UTR of 142 nt. The above mentioned 5'UTR characteristics are inhibitory for ribosomal scanning. Indeed, relative to the beta-globin 5'UTR, the complex 5'UTRs strongly repressed initiation of protein synthesis in pre-MBT Xenopus embryos. However, later in embryogenesis, the inhibition was partly relieved. The results show temporal translational control by these 5'UTRs. Transgenic embryos showed that the 5'UTRs allowed translation throughout the embryo; spatial control could not be observed. Interestingly, a fragment in the PDGF-A 5'UTR highly similar to an element in the human PDGF-A 5'UTR is complementary to Xenopus 18S ribosomal RNA. None of these Xenopus 5'UTRs contains an IRES, as determined by injecting bicistronic constructs.     

Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated

Retrotransposon L1 is a mobile genetic element of the LINE family that is extremely widespread in the mammalian genome. It encodes a dicistronic mRNA, which is exceptionally rare among eukaryotic cellular mRNAs. The extremely long and GC-rich L1 5' untranslated region (5'UTR) directs synthesis of numerous copies of RNA-binding protein ORF1p per mRNA. One could suggest that the 5'UTR of L1 mRNA contained a powerful internal ribosome entry site (IRES) element. Using transfection of cultured cells with the polyadenylated monocistronic (L1 5'UTR-Fluc) or bicistronic (Rluc-L1 5'UTR-Fluc) RNA constructs, capped or uncapped, it has been firmly established that the 5'UTR of L1 does not contain an IRES. Uncapping reduces the initiation activity of the L1 5'UTR to that of background. Moreover, the translation is inhibited by upstream AUG codons in the 5'UTR. Nevertheless, this cap-dependent initiation activity of the L1 5'UTR was unexpectedly high and resembles that of the beta-actin 5'UTR (84 nucleotides long). Strikingly, the deletion of up to 80% of the nucleotide sequence of the L1 5'UTR, with most of its stem loops, does not significantly change its translation initiation efficiency. These data can modify current ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5'UTRs and call into question the conception that every long GC-rich 5'UTR working with a high efficiency has to contain an IRES. Our data also demonstrate that the ORF2 translation initiation is not directed by internal initiation, either. It is very inefficient and presumably based on a reinitiation event.     

Localized IRES-Dependent Translation of ER Chaperone Protein mRNA in Sensory Axons

Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.     

3'UTRs of glutathione peroxidases differentially affect selenium-dependent mRNA stability and selenocysteine incorporation efficiency

Selenoprotein mRNAs are particular in several aspects. They contain a specific secondary structure in their 3'UTR, called Secis (selenocysteine inserting sequence), which is indispensable for selenocysteine incorporation, and they are degraded under selenium-limiting conditions according to their ranking in the hierarchy of selenoproteins. In the familiy of selenium-dependent glutathione peroxidases (GPx) the ranking is GI-GPx > or = PHGPx > cGPx = pGPx. This phenomenon was studied by mutually combining the coding regions of GI-GPx, PHGPx and cGPx with their 3'UTRs. HepG2 cells were stably transfected with the resulting constructs. Expression of glutathione peroxidases was estimated by activity measurement and Western blotting, the selenium-dependent mRNA stability by real-time PCR. Whereas 3'UTRs from stable PHGPx and GI-GPx could be exchanged without loss of stability, they were not able to stabilize cGPx mRNA. cGPx 3'UTR rendered GI-GPx and PHGPx mRNA unstable. Thus, cGPx mRNA contains selenium-responsive instability elements in both the translated and the untranslated region, which cannot be compensated by one of the stable homologs. Stabilizing efficiency of an individual GPx 3'UTR did not correlate with the efficiency of selenocysteine incorporation. PHGPx 3'UTR was equally effective as cGPx 3'UTR in enhancing GPx activity in all constructs, while GI-GPx 3'UTR showed a markedly lower efficacy. We conclude that different mRNA sequences and/or RNA-binding proteins might regulate mRNA stability and translation of selenoprotein mRNA.     

Post-transcriptional regulation of human cathepsin L expression

The expression of cathepsin L, a lysosomal protease, is known to be elevated in cancer and other pathologies. Multiple splice variants of human cathepsin L with variable 5'UTRs exist, which encode for the same protein. Previously we have observed that variant hCATL A (bearing the longest 5'UTR) was translated in vitro with significantly lower efficiency than variant hCATL AIII (bearing the shortest 5'UTR). Contrary to these findings, results of the present study reveal that in cancer cells, hCATL A mRNA exhibits higher translatability in spite of having lower stability than AIII. This is the first report demonstrating a highly contrasting trend in translation efficiencies of hCATL variants in rabbit reticulocytes and live cells. Expression from chimeric mRNAs containing 5'UTRs of A or AIII upstream to luciferase reporter cDNA established the A UTR to be the sole determinant for this effect. Transient transfections of bicistronic plasmids and mRNAs confirmed the presence of a functional Internal Ribosome Entry Site in this UTR. Our data suggest that differential stability and translation initiation modes mediated by the 5'UTRs of human cathepsin L variants are involved in regulating its expression.     

Rapid deadenylation and Poly(A)-dependent translational repression mediated by the Caenorhabditis elegans tra-2 3' untranslated region in Xenopus embryos

The 3' untranslated region (3'UTR) of many eukaryotic mRNAs is essential for their control during early development. Negative translational control elements in 3'UTRs regulate pattern formation, cell fate, and sex determination in a variety of organisms. tra-2 mRNA in Caenorhabditis elegans is required for female development but must be repressed to permit spermatogenesis in hermaphrodites. Translational repression of tra-2 mRNA in C. elegans is mediated by tandemly repeated elements in its 3'UTR; these elements are called TGEs (for tra-2 and GLI element). To examine the mechanism of TGE-mediated repression, we first demonstrate that TGE-mediated translational repression occurs in Xenopus embryos and that Xenopus egg extracts contain a TGE-specific binding factor. Translational repression by the TGEs requires that the mRNA possess a poly(A) tail. We show that in C. elegans, the poly(A) tail of wild-type tra-2 mRNA is shorter than that of a mutant mRNA lacking the TGEs. To determine whether TGEs regulate poly(A) length directly, synthetic tra-2 3'UTRs with and without the TGEs were injected into Xenopus embryos. We find that TGEs accelerate the rate of deadenylation and permit the last 15 adenosines to be removed from the RNA, resulting in the accumulation of fully deadenylated molecules. We conclude that TGE-mediated translational repression involves either interference with poly(A)'s function in translation and/or regulated deadenylation.     

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.     

The 3' untranslated region of bovine follicle-stimulating hormone beta messenger RNA downregulates reporter expression: involvement of AU-rich elements and transfactors

FSHbeta mRNA has a unique 3' untranslated region (3'UTR) that is highly conserved across the species. Sequence analyses of the mouse, rat, human, bovine, and ovine 3'UTRs revealed the presence of elements implicated in mRNA instability and translational control such as AU-Rich Element (ARE) and lipoxygenase differentiation control elements. Bovine FSHbeta 3'UTR down-regulated reporter expression in alphaT3-1 and NIH3T3 cells, but not in HEK 293 cells, suggesting the involvement of a cell-specific factor or mechanism. The presence of a 3'UTR did not influence reporter mRNA stability, but it did decrease its association with polysomes, indicating that the downregulatory effect may be exerted at the translational level. The segment spanning 601-800 bases (U4) of the bovine FSHbeta 3'UTR was found to be the most effective downregulating segment, its effect being equal to that of the full-length 3'UTR. RNA electrophoretic mobility shift assay with U4 showed the presence of specific transfactors in the cytosolic preparations of bovine pituitary and the cell lines. U4 contained an ARE that appeared to be functional, because the mutated U4 ARE was ineffective in downregulating the reporter expression and inhibiting [(32)P]-labeled U4-transfactor complex formation. Downregulation of reporter activity by the full-length 3'UTR and U4 could be overcome by overexpression of HuR, a protein known to stabilize ARE-containing mRNAs in NIH3T3 cells, but not in the alphaT3-1 cells, once again indicating that factors other than HuR may also be involved in the regulation of FSHbeta in the pituitary.     

Mu-opioid receptor expression in High Five insect cells is regulated by 5' untranslated region (5'UTR)

There is increasing evidence that the 5'UTR of mRNAs affects regulation of gene expression in eukaryotic cells. We examined the overexpression of the mu-opioid receptor in High Five insect cells, employing rat mu-receptor cDNA linked to variable lenghts of their native 5'UTR. The sequences employed consist of either 209 nucleotides (termed ,,long") upstream the translation initiation site of the mu-receptor mRNA, or a truncated 5'UTR comprising only 11 nucleotides (,,short"). These constructs served to generate recombinant baculovirus for the expression of mu-receptor protein in High Five insect cells. 48 hours after baculovirus infection cells were harvested for mu-receptor characterization or RNA analysis. Scatchard analysis of radioligand binding consistently revealed three to four fold higher concentrations of the mu-opioid receptors expressed with the ,,long" over the ,,short" UTR containing baculovirus. The distinct expression rates of mu-receptors paralleled the amounts of mRNAs determined by RNase protection assay. Regardless of the distinct 5'UTR regions, the expressed opioid receptors displayed identical high affinity binding characteristics for the opioid antagonist diprenorphine and similar EC50 values to inhibit forskolin (10(-5) M) stimulated cAMP synthesis. Our results demonstrate that the native 5'UTR of the mu-opioid receptor has an enhancing effect on expression in the baculovirus/insect cell system.     

Biochemical characterization of 39-kDa class I histocompatibility antigen in plasma. A secretable membrane protein derived from transmembrane domain deletion

Three human class I major histocompatibility antigens (HLA) with molecular masses of 44, 39, and 36 kDa were identified in plasma by immunoprecipitation and immunoblotting. Further biochemical characterization showed that these antigens in plasma could be fractionated by Sephacryl S-300 column chromatography into two different pools. The 44-kDa intact HLA heavy chains are detected only in pool I and have an apparent molecular weight of 200,000 as determined by calibrated gel filtration column chromatography. The 39- and 36-kDa HLA heavy chains are present only in pool II and have an apparent molecular weight of 50,000. HLA in pool I can be extracted by Triton X-114 detergent, but 39- and 36-kDa plasma HLA in pool II are water soluble and not extractable by Triton X-114. Amino acid sequences of NH2 termini for 44- and 39-kDa plasma HLA are identical to that of cellular HLA. In contrast, the NH2-terminal amino acid sequence for 36-kDa plasma HLA has not been reported previously for any other proteins. Since the loss of both transmembrane domain and cytoplasmic tail at the carboxyl terminus of HLA will generate a 36-kDa protein, the findings suggest that the 39-kDa HLA might be the product of alternatively spliced mRNA with deletion of the exon coding for transmembrane domain. By using polymerase chain reaction and DNA sequencing, the presence of alternatively spliced mRNA with deletion of the transmembrane domain exon was identified in mononuclear leukocytes of peripheral blood. This alternatively spliced HLA mRNA was not detectable in mononuclear leukocytes of an individual who had no 39-kDa plasma HLA. This finding indicates that the alternatively spliced mRNA in mononuclear leukocytes is responsible for the synthesis of a secretable class I HLA.