Untranslated Region Region Research Articles

Interleukin-1-mediated Stabilization of Mouse KC mRNA Depends on Sequences in both 5′- and 3′-Untranslated Regions

mRNA transcribed from the mouse KC chemokine gene accumulated to significantly higher levels in multiple cell types after treatment with interleukin 1alpha (IL-1alpha) as compared with tumor necrosis factor-alpha (TNFalpha). Although TNFalpha and IL-1alpha both signaled the activation of nuclear factor kappaB and enhanced transcription of the KC gene with equal potency, only IL-1alpha treatment resulted in stabilization of KC mRNA. Nucleotide sequences that confer sensitivity for IL-1alpha-mediated mRNA stabilization were identified within the 5'- and 3'-untranslated regions (UTRs) of KC mRNA using transient transfection of chimeric plasmids containing specific portions of KC mRNA linked to the chloramphenicol acetyltransferase (CAT) gene. When plasmids containing either the 3'- or 5'-UTR of KC mRNA were used, the half-life of CAT mRNA was unaltered either in untreated or IL-1alpha-stimulated cells. In contrast, CAT mRNA transcribed from plasmids that contained both the 5'- and 3'-UTRs of the KC mRNA decayed more rapidly than control CAT mRNA, and this enhanced decay was prevented in cells treated with IL-1alpha. A cluster of four overlapping AUUUA motifs within the 3'-UTR was required, whereas the 5'-UTR region exhibited orientation dependence. These findings indicate that cooperative function of the two nucleotide sequences involves a distinct signaling pathway used by IL-1alpha but not TNFalpha.

Sequence diversity in the 5′-UTR region of GB virus C/hepatitis G virus assessed using sequencing, heteroduplex mobility analysis and single-strand conformation polymorphism

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense RNA virus belonging to the Flaviviridae family identified recently. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect GBV-C/HGV RNA using nested primers designed to amplify 245 bp of the 5′-untranslated region (UTR). GBV-C/HGV RNA was detected in 20.7% of 101 HCV-RNA positive and 6.8% of 44 HCV-RNA negative specimens. Sequencing of the PCR products demonstrated they had between 84.3 and 100% nucleotide identity. Most of the diversity corresponded to two variable regions identified within the 5′-UTR. Phylogenetic analysis indicated that GBV-C/HGV subtypes present in Australia belonged to group 2 and were closest in evolutionary terms to isolates from the USA and Europe. All isolates were analysed using single-strand conformation polymorphism (SSCP) and heteroduplex mobility analysis (HMA) on 8% non-denaturing polyacrylamide gels. SSCP of the isolates identified a number of distinct conformation polymorphisms that corresponded with sequence-determined genetic diversity. HMA was developed to assess the amount of genetic diversity between isolates without the need for sequencing. The average difference between the predicted divergence of two isolates calculated from the mobility of the heteroduplex and the actual value (based on nucleotide sequence) was 2.3% in this sample of isolates, where the mean sequence divergence was 8.52%.

The 5'-untranslated region of GM-CSF mRNA suppresses translational repression mediated by the 3' adenosine-uridine-rich element and the poly(A) tail

Granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA levels are controlled post-transcriptionally by the 3'-untranslated region (UTR) adenosine-uridine-rich element (ARE). In untransformed, resting cells, the ARE targets GM-CSF mRNA for rapid degradation, thereby significantly suppressing protein expression. We used a rabbit reticulocyte lysate (RRL) cell-free system to examine translational regulation of GM-CSF expression. We uncoupled decay rates from rates of translation by programming the RRL with an excess of mRNAs. Capped, full-length, polyadenyl-ated human GM-CSF mRNA (full-length 5'-UTR AUUUA+A90) and an ARE-modified version (full-length 5'-UTR AUGUA+A90) produced identical amounts of protein. When the 5'-UTR was replaced with an irrelevant synthetic leader sequence (syn 5'-UTR), translation of syn 5'-UTR AUUUA+A90 mRNA was suppressed by >20-fold. Mutation of the ARE or removal of the poly(A) tail relieved this inhibition. Thus, in the absence of a native 5'-UTR, the ARE and poly(A) tail act in concert to block GM-CSF mRNA translation. Substitutions of different regions of the native 5'-UTR revealed that the entire sequence was essential in maintaining the highest rates of translation. However, shorter 10-12 nt contiguous 5'-UTR regions supported 50-60% of maximum translation. The 5'-UTR is highly conserved, suggesting similar regulation in multiple species and in these studies was the dominant element regulating GM-CSF mRNA translation, overriding the inhibitory effects of the ARE and the poly(A) tail.

TGF-β 1, regulation of Alzheimer amyloid precursor protein mRNA expression in a normal human astrocyte cell line: mRNA stabilization

The transforming growth factor, TGF-β 1, has been found to be increased in the central nervous system of Alzheimer's disease (AD) patients, elevates amyloid precursor protein (APP) mRNA levels in rat primary astrocytes, and may initiate or promote the deposition of amyloid-β (Aβ) peptide in AD. Excess APP production in AD, which potentially leads to amyloidogenesis, is in part due to over expression of APP mRNA. The production of APP in a normal human cell line in contrast to transformed or animal cells provides a meaningful model to study the regulation of APP gene expression by cytokines that promotes amyloidogenesis. Here, we report that TGF-β 1 treatment of human astrocytes markedly elevated APP mRNA levels, and also increased the half-life of APP message by at least five-fold. Under this condition, as detected by mobility shift and UV cross-linking analysis, a novel 68 kDa RNA–protein complex was formed, involving an 81 nucleotide (nt) fragment within the 3′-untranslated region (UTR), but not the 5′-UTR and coding region of APP mRNA. Insertion of the 3′-UTR onto the chloramphenicol acetyl transferase (CAT) mRNA conferred TGF-β 1 mediated mRNA stability in transfected human astrocytes. On the other hand, the same insert carrying a deletion of the APP mRNA cis-element fragment had no effect on CAT mRNA stability. A model of APP mRNA regulation is presented in which TGF-β 1 induced stabilization of APP message involves the binding activity of a 68 kDa RNA–protein complex within the 3′-UTR, which is likely linked to a reduction in the rate of APP mRNA decay.

Identification of 3'UTR region implicated in tau mRNA stabilization in neuronal cells.

Tau, a neuronal microtubule-associated protein (MAP) plays an important role in the formation and maintenance of neuronal polarity. Tau mRNA is a stable message and exhibits a relatively long half-life in neuronal cells. The regulation of mRNA stability is a crucial determinant in controlling mRNA steady-state levels in neuronal cells and thereby influences gene expression. The half-lives of specific mRNAs may be dependent on specific sequences located at their 3'untranslated region (UTR), which in turn, may be recognized by tissue-specific proteins. To identify the sequence elements involved in tau mRNA stabilization, selected regions of the 3'UTR were subcloned downstream to c-fos reporter mRNA or to the coding region of the tau mRNA. Using stably transfected neuronal cells, we have demonstrated that a fragment of 240 bp (H fragment) located in the 3'UTR can stabilize c-fos and tau mRNAs. Analysis of stably transfected cells indicated that the transfected tau mRNAs are associated with the microtubules of neuronal cells, suggesting that this association may play a role in tau mRNA stabilization. This step may be a prerequisite in the multistep process leading to the subcellular localization of tau mRNA in neuronal cells.

Genetic analysis of the 3′ untranslated region of the tumour necrosis factor shows a highly conserved region in rheumatoid arthritis affected and unaffected subjects

Tumour necrosis factor (TNF) is a key proinflammatory mediator in rheumatoid arthritis (RA). The TNF locus, situated in the class III region of the MHC, is flanked by five microsatellite...

Negative Control of the Poly(A)-binding Protein mRNA Translation Is Mediated by the Adenine-rich Region of Its 5′-Untranslated Region

Translation of the mRNA for the poly(A)-binding protein (PABP) may be autoregulated by the binding of PABP to the A-rich segment of its 5'-untranslated region (UTR). To test this hypothesis, we examined the effect of different fragments of the 5'-UTR from human PABP cDNA on the translation of the beta-galactosidase (beta-Gal) reporter gene. Presence of the A-rich sequence from the 5'-UTR of PABP mRNA inhibited expression of the chimeric beta-Gal gene in transfected HeLa cells. The differences in expression of beta-Gal polypeptide was due to the translational repression of beta-Gal mRNA containing the A-rich 5'-UTR of PABP mRNA. The A-rich region of the 5'-UTR located within nucleotides 58-146 of PABP mRNA was sufficient to mediate translational control of this mRNA expression. We also examined the effect of overexpression of PABP mRNA in HeLa cells. The ectopic PABP mRNA without the A-rich 5'-UTR region was translated efficiently, whereas the translation of the endogenous PABP mRNA was substantially inhibited in the transfected cells. In contrast, the ectopic PABP mRNA containing the A-rich 5'-UTR region did not show similar effect on the translation of the endogenous PABP mRNA in these cells. These results suggest that feedback control of mRNA translation is involved in regulating PABP expression in HeLa cells.

Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non-small cell lung cancers (NSCLC).

Homozygous deletions of the tumor suppressor gene p16INK4A and deficiency of methylthioadenosine phosphorylase (MTAP), both located on chromosome 9p21, have been independently reported in non-small cell lung cancer (NSCLC). To determine the frequency of co-deletion of these two genes, we investigated 50 samples of primary NSCLC using a quantitative PCR-ELISA. All specimens were fixed in formalin, paraffin embedded and stored until assayed. Histologic subtypes included 25 adenocarcinomas (50%), 21 squamous cell carcinomas (42%) and four large cell carcinomas (8%). Homozygous deletions of MTAP exon 8 could be detected in 19 of 50 NSCLC samples (38%). Adenocarcinoma (11 of 25, 44%) showed a higher deletion frequency than squamous cell carcinoma (six of 21, 29%). In contrast, homozygous p16INK4A deletions were detected in only nine of 50 (18%) samples using specific primers for p16INK4A exon 1alpha. No difference between the histological subtypes and p16INK4A deletion frequency was observed. We further investigated the ten samples with MTAP deletions but intact p16INK4A exon 1alpha with primers specific for p16INK4A exon 3, the exon nearest to MTAP exon 8. Interestingly, none of the ten samples had deletion of the p16INK4A exon 3 coding region. Fine mapping analysis performed in ten samples showed a frequent breakpoint between MTAP exon 4 and exon 5. In addition, p16 protein expression could not be detected in five out of six samples with intact p16 but deleted MTAP locus. These data show a high frequency of homozygous MTAP deletions in NSCLC which is associated with detectable co-deletion of p16INK4A in only half of the cases. This result suggests the existence either of another tumor suppressor gene telomeric of p16INK4A or of deletions involving 3'-untranslated (3'-UTR) regulatory regions of p16INK4A that can interfere with its expression or function.

Small deletions in the regulatory 3′ UTR of the human α-tropomyosin gene identified by differential display

The differential display technique (DDT) was used to compare Fanconi anaemia (FA) fibroblasts with those of normal controls in a screen for genes involved in DNA repair, recognizing and handling damage or indicating cell cycle abnormalities as a result of genetic changes. The DDT revealed two different deletions of 5 and 11 bp at a single locus in the 3′ untranslated region (UTR) of a gene known to encode human α-tropomyosin (TPM1) in FA cells. These small deletions were detected by analysis of shifted 900-bp long cDNA fragments on polyacrylamide gels. They were characterized as loss of GTTTT or TGTTTTGTTTT, respectively, in a region with five GTTTT tandem repeats. Since it was postulated that the 3′ UTR of the TPM1 gene plays a regulatory role in cell differentiation and tumour suppression, the existence and possible patterns of deletions in a variety of normal donors was investigated. The heterogenous distribution of non-deleted, 5- and 11-bp deleted 3′ UTR regions indicate a polymorphism of the TPM1 gene in this tandem repeat motif. Therefore the expression pattern of these mutations among FA and non-FA cells rendered any direct relationship to the putative DNA repair defect in FA unlikely. Of note, however, the fact remains that such deletions reportedly facilitate mRNA degradation and may bear significance in the TPM1 gene action. Finally, of further interest is the finding that even small deletions can be identified by DDT in addition to the identification of the differential expression patterns of genes.

Structure and Organization of the DrosophilaCholinergic Locus

The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein which pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation.

The 3′-end of the human β-actin gene enhances activity of the β-actin expression vector system: construction of improved vectors

The human β-actin promoter has been widely used to drive expression of genes of interest in mammalian cell lines and transgenic mice. The original form of the human β-actin expression vector contains upstream sequences, 5′UTR (untranslated region) and intron 1 from the β-actin gene linked to a three restriction site polylinker and SV40 (Simian Virus 40) 3′UTR. We have modified this vector now to contain the highly conserved β-actin 3′UTR plus flanking region which replaces the SV40 sequences. An additional modification has removed the mRNA peripheral localization sequences present in the β-actin 3′UTR. The new vectors also contain an improved polylinker. The activity of these two new vectors has been compared with that of the original vector and that of a vector using the popular cytomegalovirus (CMV) promoter. Mouse C2 myoblasts were transfected with each vector driving expression of enhanced green fluorescent protein (EGFP) and analyzed for EGFP mRNA levels. We find that both new vectors drive twice the level of mRNA accumulation of the original vector and over 30-times that of the CMV promoter. This suggests that these new vectors will provide a substantial elevation in levels of expression by virtue of inclusion of the β-actin 3′UTR plus flanking region.

First Report of Bean Common Mosaic Necrosis Potyvirus (BCMNV) Infecting Common Bean in California.

During the 1996 growing season (June to September) an outbreak of bean common mosaic was detected in a navy bean field (cv. Snow Bunting) in Colusa County, CA. Early field inspections (August 1996) revealed an incidence of 5 to 10% infection, whereas a late field inspection (September) showed an incidence of 70 to 90% infection. Enzyme-linked immunosorbent assay (ELISA) was performed on 18 leaf samples from symptomatic plants collected from this field with two monoclonal antibodies (Mab): Mab I-2, which detects bean common mosaic necrosis virus (BCMNV) strains (previously necrotic or serotype A bean common mosaic potyvirus [BCMV] strains), and Mab 197, which detects BCMV strains (previously non-necrotic or serotype B BCMV strains) and BCMNV (3). ELISA results indicated BCMNV infection in all 18 samples. In order to confirm ELISA results and to further characterize the viral isolate(s), primary leaves of the differential bean cvs. Black Turtle Soup (BTS) T-39, Topcrop, Amanda, and Sutter Pink were inoculated mechanically with sap prepared from the same leaves used for ELISA. Within 1 week, BTS T-39 and Topcrop plants showed necrotic spots on inoculated leaves and systemic necrosis and death (black root rot symptoms), Sutter Pink showed typical systemic mosaic symptoms, and Amanda showed necrotic spots and restricted vein necrosis on inoculated leaves. These reactions were consistent with infection by the NL-3 strain of BCMNV (1). Reverse transcriptase-polymerase chain reaction was used to amplify a portion of the genome of the virus that contains the 3' end of the coat protein (CP) gene and the 3' untranslated region (UTR). A DNA fragment of approximately 670 bp was amplified and DNA sequence analysis revealed that the nucleotide sequences of the 3' end of the CP and the UTR region of the California BCMNV isolate were 98 and 94% similar to those of the Michigan isolate of the BCMNV NL-3 strain (2), respectively. Together, these results suggest that the outbreak of bean common mosaic in the cv. Snow Bunting navy beans was caused by a pathogroup VI BCMNV isolate, and DNA sequence information suggests that it is similar to the NL-3 strain of BCMNV. This is the first report of BCMNV in California.

Isolation of a mRNA instability sequence that is cis-dominant to the ompA stability determinant in Escherichia coli

Transcriptional fusions with ompA and bla have been used to identify a novel mRNA instability element. A 287-nucleotide (nt) sequence containing a repetitive extragenic palindrome (REP) from the chloramphenicol acetyltransferase ( cat) gene was inserted into the 3' untranslated region (UTR) of the ompA gene. In one orientation, the insert had no effect on the half-life of the ompA-cat chimeric transcript. In the other orientation, however, the sequence functioned as a destabilizing element and was dominant to the 5′-UTR ompA and REP stability elements. The orientation-dependent effect of the instability sequence suggests that sequence rather than structure alone is important to the function of the instability determinant. In addition, the instability sequence also destabilized an ompA-bla fusion construct when fused to its 3′-UTR region. A sensitive RNA ligation/PCR amplification technique was developed and used to analyze RNA decay intermediates. The results indicated that degradation of the chimeric transcript initiated within the 287-nt inserted sequence.

Sequence of the 5'-flanking and 5'-UTR regions of the rat endothelin-A receptor gene.

Endothelin receptors bind peptides of the endothelin family, the most potent vasoconstrictors known, and have been implicated in hypertension. To begin to define DNA sequences necessary for the transcriptional regulation of the rat endothelin type A receptor (ETA), we have sequenced the 5'-untranslated region (UTR) and part of the 5'-flanking region, a total of 1153 nucleotides. Comparison of the rat and human sequences revealed a 57% similarity in the 5'-flanking sequences, and a 63% similarity in the 5'-UTRs. Several conserved sequences were identified, including GATA and E-boxes, which may be important for the regulation of ETA gene expression. Primer extension analysis identified two transcription initiation sites within the rat gene.

Degradation of unstable interleukin-1 alpha mRNA in a rabbit reticulocyte cell-free system. Localization of an instability determinant to a cluster of AUUUA motifs.

Labeled transcripts of interleukin-1 alpha (IL-1 alpha) cDNA were rapidly degraded in incubations with rabbit reticulocyte lysate (RRL). In contrast, a transcript of superoxide dismutase cDNA was stable in control incubations. A transcript of the 3'-untranslated region (UTR) of IL-1 alpha was rapidly degraded while that of the 5'-UTR and coding region was stable. This degradative activity was present in the post-ribosomal supernatant. Degradation of the 3'-UTR transcript was inhibited by the addition of a large excess of an 80-base RNA containing four AUUUA repeats, but not by the same RNA without such repeats. This suggested that AUUUA motifs were responsible for the instability of the 3'-UTR transcript. The 80-base RNA did not act as a competitive substrate for a nuclease since it was not degraded. Partial transcripts of IL-1 alpha 3'-UTR were incubated with RRL to localize instability determinants. Transcripts containing at least three clustered AUUUA motifs were rapidly degraded, while transcripts containing four scattered AUUUA motifs were stable. To study the mechanism of RNA degradation, the RRL was passed through an affinity column that retained AUUUA-binding proteins. The flow-through or the fraction eluted from such a column were inactive, but the two fractions together degraded the 3'-UTR transcript. This indicated that proteins bound by the affinity column did not have nuclease activity but targeted this RNA for degradation.

Structure of the 5' untranslated regulatory region of ferritin mRNA studied in solution

Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.

The nucleotide sequence encoding the hamster 78-kDa glucose-regulated protein (GRP78) and its conservation between hamster and rat

The complete nucleotide (nt) sequence encoding the hamster 78-kDa glucose-regulated protein has been determined usinga cDNA plasmid p3C5. Comparison of the nucleotide sequences from rat and hamster showed a strong conservation in the coding region as well as 5'- and 3 '-untranslated regions (UTRs). The relatively long (206 nt for rat) 5'UTR shares 72% sequence homology between rat and hamster in the 142 nt upstream from the ATG start codon. This conserved region contained an imperfect inverted-repeat sequence. The long 5 ' UTR region is capable of forming stable dyad structures. The homology within the rat and hamster protein-coding region is 93.7%, with most of the differences resulting in silent site mutations. Out of the 654 amino acids, only four changes are detected, two of which are located in the signal peptide. While the sizes of the 3'UTR are different between the two species compared, strong sequence homologies (95%) were observed throughout the entire UTRs. Also, the 3'UTR was not rich in A + T residues as found in other eukaryotic mRNAs.