Intron 2 of human beta-globin in 3′-untranslated region enhances expression of chimeric genes

The possibility of enhancing heterologous gene expression in mammalian cells by the introduction of an intron in 3′ untranslated region (UTR) was investigated. To this end, a fragment of human betaglobin gene with intron 2 and flanked exon regions was introduced into the vector-encoding green fluorescent protein TagGFP2 after the TagGFP2 stop-codon (Int⁺). The distance between the stop-codon and the exon junction was 35 nucleotides. It ensured that Int+ mRNA was resistant to degradation by nonsense mediated decay (NMD) machinery. A control vector Intcontained corresponding intronless sequence of the beta-globin mRNA. On the same plasmid, the second gene encoded far-red fluorescent protein Katushka was used to normalize fluorescence for transfection efficiency and expression level in individual cells. Transiently transfected HEK293T cells were analysed by flow cytometry. It was shown that cells transfected with plasmid carrying the Int+ gene possess 1.8 ± 0.2 fold higher green fluorescence compared to Intcells. The observed effect was used to enhance expression of destabilized variants of yellow fluorescent protein TurboYFP-dest with high degradation rate in mammalian cells. We believe that introduction of beta-globin intron in the 3′-UTR of the chimeric gene can be used to enhance its expression and may be advantageous in some cases when usage of 5′ UTR intron is inappropriate.     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Stability of plant mRNAs depends on the length of the 3′-untranslated region

Eukaryotic mRNAs that prematurely terminate translation are recognized and degraded by nonsense mediated decay (NMD). This degradation pathway is well studied in animal and yeast cells. The data available imply that NMD also takes place in plants. However, the molecular mechanism of recognition and degradation of plant RNAs containing premature terminator codon (PTC) is not known. Here we report that in plant cells this mechanism involves the recognition of the sizes of the 3'-untranslated regions (3'UTR). Plant 3'UTRs longer than 300 nucleotides induce mRNA instability. Contrary to mammalian and yeast cells, this destabilization does not depend on the presence of any specific sequences downstream of the terminator codon. Unlike nuclear-produced mRNAs, plant virus vector long 3'UTR-containing RNAs, which are synthesized directly in the cytoplasm, are stable and translated efficiently. This shows that RNAs produced in the cytoplasm by viral RNA-dependent RNA polymerase are able to avoid the proposed mechanism.     

Limitation in use of luciferase reporter genes for 3′-untranslated region analysis

Luciferase reporter genes are widely used for the functional characterization of regulatory elements in 3′-untranslated region (3′-UTR). Using a transient expression assay system with pancreatic cell lines, we demonstrated that luciferase reporter gene constructs show not only the elements with special sequences in 3′-UTR that can affect luciferase activity, but also elements containing random sequences that were ligated into the same site. The extent of the decrease in luciferase activity was dependent on the length of the DNA fragments. Our findings strongly suggested a need to re-examine the 3′-UTR characterizations of many eukaryotic genes which have been studied to date with luciferase reporter genes. Lintao Wang and Jingjing Zhang are equal contributors.     

Conservation of the 3′-untranslated regions of calmodulin mRNAs in cetaceans

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5- and 3-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3- untranslated region (3UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3UTR sequences. Such high conservation of the 3UTRs suggests a specialized and significant function for this region in mammals.     

One functional variant in the 3′-untranslated region of TLR4 is associated with the elevated risk of ventilator-associated pneumonia in the patients with chronic obstructive pulmonary disease

The aim of this study was to identify the association polymorphism (rs11536889) in the 3′-untranslated region (3′-UTR) of Toll-like receptors 4 (TLR4) and the risk for ventilator-associated pneumonia (VAP). miRNA database online and luciferase assays were used to validate TLR4 as the target gene of miR-1236. Enzyme-linked immunosorbent assay analysis and western blot were used to analyze the level of TLR4 in different genotype groups. In the present study, miR-1236 was predicted to bind to the rs11536889 G allele rather than the rs11536889 C allele, which was further confirmed by the luciferase activity suppressed by a fragment of 3′-UTR containing the rs11536889 G allele induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6). Bronchial epithelial cells isolated from participants genotyped as GG, GC, and CC, with no remarkable difference in TLR4 messenger RNA (mRNA) levels were observed among these genotype groups. After stimulating by LPS, a TLR4 ligand, the CC-genotyped cells expressed higher levels of IL-8, IL-6, and tumor necrosis factor alpha (TNF-α) on their surfaces than cells with the other genotypes. Finally, the western blot analysis results showed that the expression level of IL-8, IL-6, and TNF-α protein was much higher in the CC group than the GC and GG groups subsequent to stimulation by LPS, and the IL-8, IL-6, and TNF-α protein levels in the GC were grouped much lower compared with the GG group. These findings indicated the regulatory association of miR-1236 with TLR4 and the abnormal expression of TLR4 caused by the presence of rs11536889 in the 3′-UTR of mRNA, which interfere with its interaction with the miR-1236, contributing to the risk of VAP.     

Identification of association between rs1057317 polymorphism in TLR4 3′-untranslated region and the susceptibility to osteoporosis

It has been proved that the expression of TLR4 is associated with a reduced risk of osteoporosis (OP). One single-nucleotide polymorphism located within the 3′-untranslated region (3′-UTR) of TLR4 may “generate” binding site of miR-34a and thereby associated with risk of OP. Bioinformatics analysis and luciferase reporter assay were used to specify the effect of polymorphisms on the interaction between miR-34a and TLR4 gene. Western blot analysis and real-time polymerase chain reaction were used to study the expressions of miR-34a, TLR4 in different groups or cells transfected with miR-34a mimics or inhibitor. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to estimate the effect of miR-34a on the apoptosis of osteoblast. TLR4 was identified as a target of miR-34a, with negative regulatory relationship predicted. The expression levels of miR-34a was comparable with each other between CC, CA, and AA groups, and the expression levels of TLR4 was evidently lower in CC compared with GG and GC groups. Also, TLR4 level in culture osteoblast (genotyped as CC) treated with miR-34a mimics was substantially downregulated compared with scramble control, while those cells (genotyped as CC) treated with miR-34a inhibitors showed increased expression of TLR4. Additionally, the apoptosis of osteoblast genotyped as CC was decreased following transfection with miR-34a mimics, while evidently promoted subsequent to transfect with miR-34a inhibitor. The regulatory association between rs1057317 polymorphism in TLR4 3′-UTR led to an inhibitory effect on the expression of TLR4 by miR-34a, which may explain the observed association between the polymorphism and the susceptibility to OP.     

An EcoRI RFLP in the 5′ region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.