Positive and negative effects on translation of the hepatitis C virus 3' untranslated region

OBJECTIVES: To determine the role of the hepatitis C virus 3' untranslated region in viral mRNA translation in transfected cells and in cell extracts. STUDY DESIGN/METHODS: Noninfectious hepatitis C virus mini-genome RNAs with various deletions in the viral 3' untranslated region were transfected into cells or translated in vitro, and the translation efficiency was determined. RESULTS: We have found that the presence of the hepatitis C virus 3' untranslated region modestly increases mRNA translation. The positive effect correlated with the binding of a 45-kDa cytoplasmic factor to the hepatitis C virus 3' untranslated region. Furthermore, the U-rich sequence in the hepatitis C virus 3' untranslated region inhibits translation of capped and polyadenylated mRNAs as a result of the hybridization. CONCLUSIONS: The modest effect of the hepatitis C virus 3' untranslated region on translation suggests that it does not play a major role in mRNA translation. The inhibitory effect of the hepatitis C virus 3' untranslated region on translation of polyadenylated mRNAs supports the notion that translation of hepatitis C virus mRNAs occurs independently of a polyA tail.     

Functional analysis of polymorphic variation within the promoter and 5' untranslated region of the neurofibromatosis type 1 (NF1) gene

The regulatory regions of the neurofibromatosis type 1 (NF1) gene have scarcely been screened either for mutations of potential pathological importance or for functional polymorphisms. To address this question, a 987 bp sequence spanning the promoter and 5' flanking sequence of the human NF1 gene was screened for sequence variants in 570 unrelated NF1 patients and 105 controls. Five novel sequence variants were identified, comprising a 14 bp deletion at -142 within the promoter region, three single nucleotide substitutions in the 5'UTR (C + 247T, C + 261G, G + 462C), and a substitution (C + 514T) at the 5' end of the coding region that served to generate a Stop codon. The latter is likely to be of pathological significance since it is predicted to lead to the synthesis of a truncated protein. The functional significance of three of the other variants (14 bp del, C + 261G, G + 462C) was explored by luciferase reporter gene expression and electrophoretic mobility shift assays. The del14 variant demonstrated allele-specific protein binding without altered reporter gene expression and the G + 462C allele showed slightly decreased reporter gene expression.     

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 markers. It has previously been shown that this region influences susceptibility to RA; two TNF microsatellite haplotypes were found to be associated with RA. Evidence from murine studies has indicated that variation in the TNF 3' untranslated region (UTR) could be associated with altered regulation of TNF biosynthesis. In order to identify possible RA associated polymorphisms, more than 800 bp of the TNF 3' UTR was genetically analysed in RA affected and unaffected subjects possessing specific RA and non-RA associated TNF microsatellite haplotypes. The TNF 3' UTR region was analysed using two mutation detection methods, PCR-SSCP and NIRCA analysis and DNA sequencing. No genetic differences were observed in the human TNF 3' UTR between subjects, that is, irrespective of RA status or TNF haplotype, and also compared with previously published TNF sequences from human sources. Therefore it can be concluded that the TNF 3' UTR in this population was highly conserved and did not influence susceptibility to RA.     

Sequence and polyadenylation site determination of the murine immunoglobulin gamma 2a membrane 3' untranslated region

We have determined the nucleotide sequence of the murine immunoglobulin gamma 2a membrane 3' untranslated region (1413 nucleotides) and approximately 679 nucleotides of downstream sequence. Two AATAAA hexanucleotide sequences are present in the 2092 nucleotide interval. The first one functions as the major polyA signal, directing cleavage and polyadenylation at a site 20 nucleotides downstream. Within 41 nucleotides downstream of the major membrane polyA signal are two sequences with 75% homology to the consensus sequence, (C/T)GTGTT(C/T)(C/T), identified by McLauchlan et al. [Nucl. Acids Res. 13, 1347-1365 (1985)]. An 80% homology match to the Berget consensus sequence, CA(C/T)TG, begins five nucleotides 3' of the major polyA site (used 20 times more than the second, downstream polyA site) [Berget Nature 309, 179-182 (1984)]. The second AATAAA, located 73 nucleotides 3' of the first, directs cleavage and polyadenylation 18 nucleotides downstream at a minor polyA site. One match with 75% homology to the McLauchlan consensus sequence begins 17 nucleotides 3' of the second (weaker) polyA site. No matches to the Berget consensus sequence are located near this second, weaker polyA site.     

Analysis on the association of human BLYS (BAFF,TNFSF13B) polymorphisms with systemic lupus erythematosus and rheumatoid arthritis

Recent studies indicated a substantial role of BLyS (BAFF, TNFSF13B) in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in humans and in animal models. This study was conducted to screen for polymorphisms of human BLYS, and to examine whether they are involved in the genetic susceptibility to human SLE and RA. A systematic polymorphism screening was performed in the coding region, 5' and 3' untranslated regions, and promoter region of human BLYS. Association of the detected polymorphisms with SLE and RA was analyzed in 221 Japanese patients with RA, 156 with SLE, and 227 healthy individuals, using the case-control approach. Four single nucleotide polymorphisms (SNPs) in the promoter, one SNP in intron 1, and one rare nonsynonymous substitution (Ala105Thr) in the coding region were detected. The BLYS SNPs were found to form three common haplotypes. Significant association with the susceptibility to SLE or RA was not observed. However, a tendency for the increase of -871T/T genotype was observed in SLE patients with anti-Sm antibody (P=0.082). BLYS mRNA level was significantly elevated in the monocytes from individuals carrying -871T (P=0.010). In addition, although statistically not significant, 105Thr allele was slightly increased in patients with RA compared with controls (P=0.058). Characterizing the functional and clinical significance of these new SNPs requires further study.     

TCRζ mRNA splice variant forms observed in the peripheral blood T cells from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology. Tyrosine phosphorylation and protein expression of the T-cell receptor ζ chain (ζ) have been reported to be significantly decreased in SLE T cells. In addition, ζ mRNA with alternatively spliced 3′ untranslated region (ζmRNA/as-3′UTR) is detected predominantly in SLE T cells, and aberrant ζ mRNA accompanied by the mutations in the open reading frame including ζ mRNA lacking exon7 (ζmRNA/exon7-) is observed in SLE T cells. These ζ mRNA splice variant forms exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface due to the instability of ζ mRNA splice variant forms as well as the reduction in interleukin (IL)-2 production after stimulating with anti-CD3 antibody. Data from cDNA microarray showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in the MA5.8 cells transfected with the ζ mRNA splice variant forms. Another 16 genes were up-regulated and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus-receptor-related 2, syndecan-1, and granzyme A.     

Roles of promoter and 3' untranslated motifs in expression of the human C5a receptor

The C5a receptor (C5aR) is a 7 transmembrane G-protein coupled receptor (GPCR) that mediates the powerful pro-inflammatory effect of the complement activation product C5a. Excess C5a generated under pathological conditions has been implicated in a variety of conditions including sepsis, asthma and rheumatoid arthritis, but very little is known about the regulation of expression of the C5aR. The 5' promoter region and 3' untranslated region (UTR) of the C5aR mRNA were cloned, generating enhanced green fluorescent protein (EGFP)-reporter plasmids, which were transfected into the monocytic cell line U937. Most of the cloned 2kb 5' region was dispensable for the expression of the reporter constructs and the majority of regulatory sequences are in the first 200 bp. Three motifs, a NFκB, a CCAAT and a NFAT site, were identified to be of importance by site directed mutagenesis for basal expression. Analysis of the 3'UTR of the C5aR mRNA showed that it contained two AU-rich elements (AREs), however site directed mutagenesis showed that these had no effect on basal expression. While the phorbol ester PMA and dibutyryl cAMP increased C5aR protein expression, these agents had no effect on the regulation of expression via the promoter or the 3'UTR. This is the first study to investigate the role of both the promoter and 3'UTR in regulating C5aR expression and our results show that regulation of the human C5aR is similar but not identical to that of the mouse C5aR.     

Altered expression of the T cell receptor-CD3 complex in systemic lupus erythematosus

SLE T cells may play a key role in autoantibody production in SLE B cells. In addition, accumulating evidence has shown that SLE T cells participate in the attack on target cells or tissues through the overproduction of pro-inflammatory cytokines or an increase in cell-to-cell adhesion. Thus, the functional abnormality of SLE T cells appears to be pivotal to an understanding of SLE pathogenesis. Accumulating evidence suggests that potential defects may reside in the proximal signal transduction around the TCR-CD3 complex. We have demonstrated that the expression of TCR zeta chain is significantly decreased in peripheral blood T cells from SLE patients. To explore the mechanism of defective expression of TCR zeta chain, we examined mRNA of TCR zeta, and found that two alternatively spliced variants such as exon 7 (-) and short 3'-UTR are detected in SLE. We review the possible role of the TCR zeta defects in autoimmunity and discuss how the splicing variants lead to downregulated protein expression of TCR zeta chain.     

TCRzeta mRNA splice variant forms observed in the peripheral blood T cells from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology. Tyrosine phosphorylation and protein expression of the T-cell receptor ζ chain (ζ) have been reported to be significantly decreased in SLE T cells. In addition, ζ mRNA with alternatively spliced 3′ untranslated region (ζmRNA/as-3′UTR) is detected predominantly in SLE T cells, and aberrant ζ mRNA accompanied by the mutations in the open reading frame including ζ mRNA lacking exon7 (ζmRNA/exon7-) is observed in SLE T cells. These ζ mRNA splice variant forms exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface due to the instability of ζ mRNA splice variant forms as well as the reduction in interleukin (IL)-2 production after stimulating with anti-CD3 antibody. Data from cDNA microarray showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in the MA5.8 cells transfected with the ζ mRNA splice variant forms. Another 16 genes were up-regulated and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus-receptor-related 2, syndecan-1, and granzyme A.     

Enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain

An essential step for a productive infection by the dengue flavivirus (DEN) is translation of the m(7)G-capped, nonpolyadenylated positive-sense RNA genome. We have recently identified sequences within the DEN 3' untranslated region (UTR) that modulate viral translation. Here, we show that the DEN type 2 (DEN2) 3'UTR stimulated translation of m(7)G-capped DEN2 5'UTR-containing reporter mRNAs in baby hamster kidney (BHK) cells compared to a 3' vector sequence. Analogous to the 3' poly(A) tail, the DEN2 3'UTR also enhanced translation of reporter mRNAs containing (i) a nonfunctional A cap, (ii) the 5'UTR of human beta-globin, or (iii) a viral internal ribosome entry site (IRES). In all cases, approximately half of the translation efficiency was due to the terminal 3' stem-loop (3'SL) domain. In addition, the 3'SL domain increased the association of mRNAs with polysomes. Together, these results indicate that the DEN2 3'UTR, mediated in part by the 3'SL domain, enhances translation initiation, possibly after recognition of the 5' cap structure.     

Father-to-daughter transmission of Cornelia de Lange syndrome caused by a mutation in the 5' untranslated region of the NIPBL Gene

Cornelia de Lange syndrome (CdLS; also called Brachmann de Lange syndrome) is a developmental disorder characterized by typical facial dysmorphism, growth and mental retardation, microcephaly, and various malformations. Mutations in the NIPBL gene have been identified in approximately 40% of reported cases, suggesting either genetic heterogeneity or that some NIPBL mutations are not detected by current screening strategies. We screened a cohort of 21 patients with no previously identified NIPBL anomaly for mutations in the 5' untranslated region (5'UTR) and the proximal promoter of the NIPBL gene. We identified a heterozygous deletion-insertion mutation in exon 1, 321 nucleotides upstream of the translation initiation codon (c.-321_-320delCCinsA) in one affected girl and her mildly affected father. This mutation altered highly conserved nucleotides, was not found in 400 control alleles, arose de novo in the father, and cosegregated with the disease in the family. Using real-time quantitative PCR, we showed that NIPBL mRNA expression was lowered in patients' lymphocytes compared to control samples. Finally, we showed that, when subcloned into a luciferase reporter vector, the mutation leads to a significant reduction of reporter gene activity. Our results demonstrate that mutations in the 5' noncoding region of the NIPBL gene can be involved in the pathogenesis of CdLS. Mutations affecting this region of the gene might be associated with a milder phenotype.     

Efficient translation of mRNAs in influenza A virus-infected cells is independent of the viral 5' untranslated region.

We test the hypothesis that the translation machinery in cells infected by influenza A virus efficiently translates only mRNAs that possess the influenza viral 5' untranslated region (5'-UTR) by introducing mRNAs directly into the cytoplasm of infected cells. This strategy avoids effects due to the inhibition of the nuclear export of cellular mRNAs mediated by the viral NS1 protein. In one approach, we transfect in vitro synthesized mRNAs into infected cells and demonstrate that these mRNAs are efficiently translated whether or not they possess the influenza viral 5'-UTR. In the second approach, an mRNA is synthesized endogenously in the cytoplasm of influenza A virus infected cells by a constitutively expressed T7 RNA polymerase. Although this mRNA is uncapped and lacks the influenza viral 5'-UTR sequence, it is efficiently translated in infected cells via an internal ribosome entry site. We conclude that the translation machinery in influenza A virus infected cells is capable of efficiently translating all mRNAs and that the switch from cellular to virus-specific protein synthesis that occurs during infection results from other processes.