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.     

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.     

Differential regulation of oestrogen receptor β isoforms by 5' untranslated regions in cancer

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs - ERβ- are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5' untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5'UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5'UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.     

Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR

A second gene encoding a functional tryptophan hydroxylase activity has recently been described (TPH2), which is expressed abundantly in brainstem, the primary site of serotonergic neurons in the CNS. As serotonin (5-HT) has an important role as a precursor of the nocturnal synthesis of the pineal gland hormone, melatonin, it was of interest to determine the relative expression of TPH1 and 2 mRNA in the rat pineal during the light:dark (L:D) cycle using sensitive real-time RT-PCR assays which were developed for each TPH isoform. TPH1 mRNA expression was 105-fold more abundant in rat pineal than TPH2, and showed a significant approximately 4-fold nocturnal increase in expression which may contribute to the previously described nocturnal increase in pineal tryptophan hydroxylase activity. TPH2 expression within the gland showed no significant variation with time of day and was very low (approximately 300 copies/gland) indicating expression in the small proportion of "non-pinealocyte" cells in the gland.     

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.     

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.     

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.     

Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E

We investigated the possibility that insulin could stimulate translation of ornithine decarboxylase (ODC) mRNA in a murine fibroblast cell line that expresses large numbers of human insulin receptors (HIR 3.5 cells). Within 3 h after exposure to 70 nM insulin, ODC enzyme activity increased approximately 50-fold and mRNA accumulation 3-fold in the HIR 3.5 cells but not in normal fibroblasts. Pretreatment of cells with cycloheximide completely inhibited insulin-stimulated ODC expression; actinomycin D partially inhibited this effect. To determine the influence of the 5' untranslated region (5'UTR) of ODC mRNA on insulin-regulated ODC expression, plasmids were constructed which contained sequences from the 5'UTR of a rat ODC mRNA interposed between the ferritin promoter and the coding region of the human growth hormone gene. These constructions were then expressed transiently in HIR 3.5 cells. Insulin stimulated a 2-4-fold change in growth hormone accumulation in the medium of cells transiently expressing plasmids containing the entire 5'UTR of ODC mRNA or just the 5'-most 115 bases, a G/C-rich conserved sequence predicted to form a stem-loop structure and shown previously to be responsible for constitutive inhibition of translation. There was a direct correlation between the extent of insulin stimulation and the predicted secondary structure of the added 5'UTR sequences. To determine whether this effect might be due to insulin activation of initiation factors responsible for melting mRNA secondary structure, we examined the effect of insulin on the phosphorylation states of two such factors, eucaryotic initiation factors eIF-4B and eIF-4E. Insulin stimulated the phosphorylation of both initiation factors; this stimulation was evident at 15 min and maximal by 60 min. These results suggest a potential general mechanism by which insulin could preferentially stimulate translation of mRNAs whose 5'UTRs exhibit significant secondary structure by activating initiation factors involved in melting such secondary structures.