Growth factor-mediated stabilization of amyloid precursor protein mRNA is mediated by a conserved 29-nucleotide sequence in the 3'-untranslated region

Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5'-untranslated region (UTR) and coding region of beta-globin and the entire 3'-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3'-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t 1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t 1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t 1/2 = 40 min). We therefore conclude that the 29-nucleotide 3'-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.     

Translation efficiency of zein mRNA is reduced by hybrid formation between the 5'- and 3'-untranslated region

The secondary structure of zein mRNA affects its translation potential. Here we show that in a cell-free system the translation efficiency of zein mRNA containing inverted repeats in the 5'- and 3'-untranslated regions is reduced. This translational block is released after deletion of the 3'-inverted repeat. We conclude that the translational block is caused by hybrid formation between the two inverted repeats. The translational efficiency of zein mRNAs, is also affected by varying the length or the primary structure of the 5'-untranslated region.     

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.     

Cis-acting sequences in the 5'-untranslated region of the ribosomal protein A1 mRNA mediate its translational regulation during early embryogenesis of Drosophila.

The rate of ribosomal (r)-protein synthesis in the early Drosophila embryo is low despite the presence of abundant, maternally supplied r-protein mRNAs. This low rate is due to specific repression of r-protein mRNA translation. In contrast to r-protein mRNAs, most other mRNAs are efficiently translated in the early embryo. Here we report on the identification of cis-acting sequences that mediate translational repression of the r-protein A1 (rpA1) mRNA. Chimeric genes containing sequences from the translationally regulated rpA1 mRNA fused to the constitutively translated alpha-tubulin mRNA were constructed and transformed into the Drosophila germ line. Translation of the corresponding hybrid mRNAs was measured in ovaries and embryos of the transgenic flies. The results indicated that a 89-nucleotide sequence in the untranslated rpA1 mRNA leader is by itself sufficient to confer full translational regulation to a heterologous mRNA.     

Specific protein binding to a conserved region of the ornithine decarboxylase mRNA 5'-untranslated region.

An RNA gel retardation assay was used to identify one or more cellular protein(s) (ornithine decarboxylase mRNA 5'-UTR binding protein (ODCBP)) that bind specifically to a conserved region of the 5'-untranslated region (5'-UTR) of rat ornithine decarboxylase (ODC) mRNA. Ultraviolet light cross-linking demonstrated that this protein has an apparent Mr = 58,000 in mammalian cells. Treatment with the oxidizing agent diamide prevented binding of the ODCBP to ODC mRNA; addition of beta-mercaptoethanol reversed this inhibition and permitted mRNA.ODCBP complex formation. Cytoplasmic extracts from a variety of animal cells and tissues demonstrated similar binding activities; however, there was marked tissue-specific expression of the protein in the rat, with brain, heart, lung, and testis containing large amounts, and kidney, spleen, and skeletal muscle expressing negligible amounts. Binding was completely prevented by several mutations within a highly conserved heptanucleotide region (CCAU/ACUC) that was within 61 bases of the initiation codon in ODC mRNAs from mammals, Xenopus, and Caenorhabditis elegans; mutations 5' and 3' of the conserved heptanucleotide domain had no effect on binding activity. Binding was not affected by manipulation of cellular polyamine levels or by treatment of cells with agents that stimulate ODC biosynthesis. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 5'-UTR of ODC mRNA from many animal species; functional consequences of this binding remain to be determined.     

Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display

The 5′-untranslated region (5′-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5′-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5′-UTRs with high translation efficiency using a ribosome display technique. A 5′-UTR random library, comprised of 5′-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5′-UTR with high translation efficiency was obtained from random 5′-UTR library.     

Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.     

A new species of enkephalin precursor mRNA with a distinct 5'-untranslated region in haploid germ cells

To elucidate the primary structure of preproenkephalin (A) mRNA expressed by haploid germ cells (round spermatids) in rat testis, we have screened a lambda gt11 cDNA library for preproenkephalin cDNA inserts. The largest cDNA insert contained a protein-coding sequence encoding 269 amino acid residues as well as 327 and 309 bases of the 5'- and 3'-untranslated regions, respectively. The protein-coding region plus 3'-untranslated region of the mRNA was over 99% homologous to that of brain preproenkephalin mRNA, whereas the 5'-untranslated region contained a distinct sequence including a partial sequence of intron A of the preproenkephalin gene [(1984) J. Biol. Chem. 259, 14301-14308; (1984) J. Biol. Chem. 259, 14309-14313]. Northern blot analysis using a 5'-end-specific probe showed that this type of preproenkephalin mRNA exists exclusively in the germ cells.     

Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein

Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.