UTR Domain Research Articles

Human pegivirus viremia in HCV/HIV co-infected patients: Direct acting antivirals exert anti-pegivirus effects.

Human pegivirus (HPgV) is a single-stranded RNA virus​ thatis closely related to hepatitis C virus (HCV)​. HPgVhasalsobeen shown to infect patients with human immunodeficiency virus (HIV). The mechanisms and disease outcomes of HPgV infections are largely unknown, although it has been implicated in both cancer and neurological diseases. There are no established therapies for HPgV. To estimate the prevalence of HPgV in a cohort of HCV/HIV co-infected patients undergoing treatment for HCV with direct acting antivirals (DAA) and investigate the effectof DAA therapy on HPgV infection. RNA was extracted from plasma samples collected at time points before, during, and after DAA. HPgV RNA abundance was quantified by droplet digital PCR assays targeting theNS5Aand 5'UTR domains and confirmed by RT-qPCR. Clinical, demographic and treatment data were analysed. HPgV RNA was detected and quantified in 26 of 100 patients' plasma (26%) before starting DAA. Patients with detectable HPgV were more likely to be male, had higher peak HIV plasma levels, and a history of injection drug use. Patients receiving sofosbuvir/ledipasvir (n=9) displayed significantly lower HPgV levels at time of DAA completion and had lower post-DAA HPgV rebound​ levels compared to patients receiving sofosbuvir/velpatasvir (n=11) although both regimens significantly reduced viremia directly following DAA completion. Sustained suppression of HPgV was​also observed among patients (n=2) receiving pegylated-interferon. HPgVRNA ​wasfrequently detected in HCV/HIV co-infected patients and ​was​supressed by DAA and pegylated interferon therapies with sofosbuvir-ledipasvir showing greatest antiviral activity. These findings suggest potential treatment strategies for HPgV infections​.

Global 5'-UTR RNA structure regulates translation of a SERPINA1 mRNA.

SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5′-untranslated region (5′-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5′-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5′-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5′-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5′-UTR structure. However, those mutations that disrupted the helix that anchors the 5′-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5′-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5′-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.

Aberrantly reduced expression of miR-342-5p contributes to CCND1-associated chronic myeloid leukemia progression and imatinib resistance

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with the Philadelphia chromosome, and the current standard of care is the use of tyrosine kinase inhibitors (TKI). However, some patients will not achieve a molecular response and may progress to blast crisis, and the underlying mechanisms remain to be clarified. In this study, next-generation sequencing was used to explore endogenous miRNAs in CML patients versus healthy volunteers, and miR-342-5p was identified as the primary target. We found that miR-342-5p was downregulated in CML patients and had a significant inhibitory effect on cell proliferation in CML. Through a luciferase reporter system, miR-342-5p was reported to target the 3’-UTR domain of CCND1 and downregulated its expression. Furthermore, overexpression of miR-342-5p enhanced imatinib-induced DNA double-strand breaks and apoptosis. Finally, by analyzing clinical databases, we further confirmed that miR-342-5p was associated with predicted molecular responses in CML patients. In conclusion, we found that both in vivo and in vitro experiments and database cohorts showed that miR-342-5p plays a key role in CML patients, indicating that miR-342-5p may be a potential target for future CML treatment or prognostic evaluation.

MiR-31-5p Promotes Oxidative Stress and Vascular Smooth Muscle Cell Migration in Spontaneously Hypertensive Rats via Inhibiting FNDC5 Expression.

Oxidative stress and the migration of vascular smooth muscle cells (VSMCs) are important for vascular remodeling in a variety of vascular diseases. miR-31-5p promotes cell migration in colorectal cancer cells but inhibits cell migration in renal cell carcinoma. However, whether miR-31-5p is involved in oxidative stress and VSMC migration remains unknown. This study shows the crucial roles of miR-31-5p in oxidative stress and VSMC migration, as well as underlying mechanisms. Experiments were carried out in primary VSMCs from aortic media of Wistar–Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), as well as the A7r5 cell line. Oxidative stress was assessed by NADPH oxidase (NOX) expression, NOX activity, and reactive oxygen species (ROS) production. Cell migration was evaluated with a Boyden chamber assay and a wound healing assay. The miR-31-5p mimic and inhibitor promoted and attenuated oxidative stress and cell migration in the VSMCs of SHR, respectively. A dual-luciferase reporter assay indicated that miR-31-5p targeted the 3’UTR domain of FNDC5. The miR-31-5p level was raised and FNDC5 expression was reduced in the VSMCs of SHR compared with those of WKY. The miR-31-5p mimic reduced FNDC5 expression in the A7r5 cells and the VSMCs of both WKY and SHR, while the miR-31-5p inhibitor only increased FNDC5 expression in the VSMCs of SHR. Exogenous FNDC5 attenuated not only the oxidative stress and VSMC migration in SHR but also the roles of the miR-31-5p mimic in inducing oxidative stress and VSMC migration. These results indicate that miR-31-5p promotes oxidative stress and VSMC migration in SHR via inhibiting FNDC5 expression. The increased miR-31-5p and reduced FNDC5 in the VSMCs of SHR contribute to enhanced oxidative stress and cell migration.

MiR-4521-FAM129A axial regulation on ccRCC progression through TIMP-1/MMP2/MMP9 and MDM2/p53/Bcl2/Bax pathways

Clear cell renal cell carcinoma (ccRCC) is the most aggressive RCC subtype with high metastasis, chemotherapy and radiotherapy resistance, and poor prognosis. This study attempted to establish the deregulations of miR-4521 and FAM129A together with their correlation to and mechanism of regulation of ccRCC development and progression. FAM129A acted as tumor promotor and miR-4521 acted as a suppressor in ccRCC. As measured in surgical tumorous tissues from ccRCC patients, FAM129A overexpression and miR-4521 deficiency together contributed to ccRCC progression by promoting advances in patients’ TNM stage and Fuhrman grade. Both the FAM129A knockdown and miR-4521 overexpression could reduce the in vitro migration and invasion abilities of renal cancer cells 786-O and ACHN, through the TIMP-1/MMP2/MMP9 pathway and could decrease their proliferation by promoting their apoptosis through the MDM2/p53/Bcl2/Bax pathway. By directly targeting the 3′-UTR domain of FAM129A, miR-4521 was negatively correlated with FAM129A/FAM129A levels in ccRCC progression and renal cancer cell malignancies. This work establishes the miR-4521-FAM129A axial regulation mechanism in ccRCC. Micro-4521 deficiency leads to FAM129A/FAM129A upregulation, which synergistically enhances the migration and invasion of renal cancer cells due to the induced decrease of TIMP-1 and increases of MMP2 and MMP9, and increases their growth through escaping apoptosis by suppressing p53 by way of upregulation of induced MDM2. The current work provides new clues to assist fundamental research into the diagnosis and treatment of ccRCC.

MicroRNAs as predictive biomarkers for myocardial injury in aged mice following myocardial infarction.

The occurrence of myocardial infarction (MI) increases appreciably with age. In the Framingham Heart Study, the incidence of MI more than doubles for men and increases more than five-fold in women (ages 55-64 years compared to 85-94 years). MicroRNAs (miRNAs) quantitatively regulate their target's expression post-transcriptionally by either silencing action through binding at the 3'UTR domains or degrading the messages at their coding regions. In either case, these regulations affect the cardiac transcriptional output and cardiac function. Among the known cardiac associated miRNA, miRNA-1, miRNA-133a, and miRNA-34a have been shown to induce adverse structural remodeling to impair cardiac contractile function. In the present study, an in vivo model of MI in young (3 month) and old (22 month) mice is used to investigate the possible role whereby these three miRNAs exert negative effects on heart function following MI. Herein we demonstrate that in older mouse heart, all three microRNAs show increased levels of expression, while miRNA-1 shows a further increase in old mouse heart following MI, which corresponds to left ventricular (LV) wall thinning. These structural changes in cardiac tissue may causes downstream LV dilation and subsequent LV dysfunction. Results presented here suggest that significantly elevated levels of miRNA-1 in post-MI old heart could be predictive of cardiac injury in older mice as the high risk biomarker for MI in older individuals.

MiR-9 Regulates the Expression of BACE1 in Dementia Induced by Chronic Brain Hypoperfusion in Rats

Background/Aims: MicroRNA-9 (miR-9) plays important roles in nervous system diseases such as glioblastoma and neurodegenerative disorders. However, how miR-9 contributes to dementia requires further study. In this study, we evaluated the role of miR-9 in dementia and the molecular mechanisms underlying its effects. Methods: A rat model of dementia was created by occlusion of the bilateral common carotid artery (2VO) for 8 weeks. Learning and memory were assessed using the Morris Water Maze (MWM). MicroRNA expression profiling was performed according to a protocol provided by LC Sciences, and quantitative real-time PCR (qRT-PCR) was used to detect the level of miR-9. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining were used to assess pathological changes in brain tissue. Western blot and immunofluorescence were employed to detect the expression of β-site APP cleaving enzyme 1 (BACE1) and c-AMP response element-binding protein (CREB). Results: Learning and memory were significantly impaired in 2VO rats, and these changes were accompanied by neuronal loss and glial activation in brain tissues. miR-9 was greatly upregulated in both the hippocampus and cortex of rats following 2VO. Knockdown of endogenous miR-9 via lentiviral vector-mediated delivery of its antisense molecule (lenti-pre-AMO-miR-9) reduced the vulnerability to dementia, reversed the increase in BACE1 expression, and ameliorated the reduction in CREB expression triggered by 2VO. BACE1 protein levels were significantly increased, but CREB protein levels were significantly decreased in the presence of miR-9 in cultured neonatal rat neurons (NRNs). AMO-miR-9 rescued the upregulation of BACE1 and downregulation of CREB elicited by miR-9 in rats. Dual luciferase assay experiments showed that overexpression of miR-9 inhibited the expression of CREB by targeting its 3’UTR domain. CREB protein was downregulated by miR-9 overexpression which was reversed by miR-9 inhibition in cultured NRNs. TEM imaging showed that miR-9 caused damage to NRNs, which was reversed by addition of AMO-miR-9. Conclusion: We conclude that miR-9 plays an important role in regulating the process of dementia induced by 2VO in rats by increasing BACE1 expression via downregulation of CREB.

Abstract B41: Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity

Abstract Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we previously reported that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins in human MDA-MB-231 TNBC cells, instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of the miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by the miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and the miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3′UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knockdown miR-17-5p reduced PDCD4 and PTEN protein expression, instead of raising them. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells also reduced PDCD4 and PTEN protein levels. To test the hypothesis that miR-17-3p passenger strand can also modulate the same mRNA targets as miR-17-5p, we performed luciferase assays using reporter constructs harboring predicted miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs. We found that the miR-17-5p mimic lowered the expression of luciferase vectors containing one predicted binding site from the PDCD4 or PTEN 3′UTR domains. However, the miR-17-3p mimic lowered the expression of luciferase vectors containing all four 3′UTR target sites of PDCD4, and two of the six predicted PTEN 3′UTR target sites. In addition, we used luciferase assays to test the hypothesis that antisense DNA-LNA against miR-17-5p mimicked passenger strand miR-17-3p. We found that anti-miR-17-5p DNA-LNA lowered the expression of luciferase reporter vectors containing two of the four predicted PDCD4 3′UTR target sites for miR-17-3p, and one of the six predicted PTEN 3′UTR target sites for miR-17-3p. From these results, we concluded that the miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs, just like the miR-17-5p guide strand. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked the miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. Supported by the Department of Biochemistry and Molecular Biology. Conflict of interest: pending PCT/US2015/015681 patent application licensed to Nikita RNA LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B41.

Non-genotype-specific role of the hepatitis C virus 5′ untranslated region in virus production and in inhibition by interferon

The 5′ untranslated region (5′UTR) of hepatitis C virus (HCV) is structured into four domains (I–IV) with numerous genotype-specific nucleotides. It is unknown whether the polymorphisms confer genotype-specific functions to the 5′UTR. Using viable JFH1-based Core-NS2 recombinants, we developed and characterized HCV genotypes 1–7 recombinants with highly diverse 5′UTRs (genotypes 1a and 3a), 2a recombinants (J6/JFH1) with 5′UTR of genotypes 1–6 or with heterotypic chimeric (1a/3a and 3a/1a) 5′UTR domains I, II or III, and 1a recombinants with 5′UTR domain I of genotypes 1–6. All were fully functional in Huh7.5 cells; therefore, the 5′UTR apparently functions in a non-genotype-specific manner in HCV production in vitro. However, adenine at the 5′-terminus was required. We demonstrated that J6/JFH1 with 5′UTR of genotypes 1–6 responded similarly to interferon-α2b. This study provides novel insight into the role of the 5′UTR in the HCV life cycle and facilitates HCV basic research and testing of 5′UTR-targeting antivirals.

The Enhancer of split complex arose prior to the diversification of schizophoran flies and is strongly conserved between Drosophila and stalk-eyed flies (Diopsidae)

BackgroundIn Drosophila, the Enhancer of split complex (E(spl)-C) comprises 11 bHLH and Bearded genes that function during Notch signaling to repress proneural identity in the developing peripheral nervous system. Comparison with other insects indicates that the basal state for Diptera is a single bHLH and Bearded homolog and that the expansion of the gene complex occurred in the lineage leading to Drosophila. However, comparative genomic data from other fly species that would elucidate the origin and sequence of gene duplication for the complex is lacking. Therefore, in order to examine the evolutionary history of the complex within Diptera, we reconstructed, using several fosmid clones, the entire E(spl)-complex in the stalk-eyed fly, Teleopsis dalmanni and collected additional homologs of E(spl)-C genes from searches of dipteran EST databases and the Glossina morsitans genome assembly.ResultsComparison of the Teleopsis E(spl)-C gene organization with Drosophila indicates complete conservation in gene number and orientation between the species except that T. dalmanni contains a duplicated copy of E(spl)m5 that is not present in Drosophila. Phylogenetic analysis of E(spl)-complex bHLH and Bearded genes for several dipteran species clearly demonstrates that all members of the complex were present prior to the diversification of schizophoran flies. Comparison of upstream regulatory elements and 3' UTR domains between the species also reveals strong conservation for many of the genes and identifies several novel characteristics of E(spl)-C regulatory evolution including the discovery of a previously unidentified, highly conserved SPS+A domain between E(spl)mγ and E(spl)mβ.ConclusionIdentifying the phylogenetic origin of E(spl)-C genes and their associated regulatory DNA is essential to understanding the functional significance of this well-studied gene complex. Results from this study provide numerous insights into the evolutionary history of the complex and will help refine the focus of studies examining the adaptive consequences of this gene expansion.

Non-Coding Changes Cause Sex-Specific Wing Size Differences between Closely Related Species of Nasonia

The genetic basis of morphological differences among species is still poorly understood. We investigated the genetic basis of sex-specific differences in wing size between two closely related species of Nasonia by positional cloning a major male-specific locus, wing-size1 (ws1). Male wing size increases by 45% through cell size and cell number changes when the ws1 allele from N. giraulti is backcrossed into a N. vitripennis genetic background. A positional cloning approach was used to fine-scale map the ws1 locus to a 13.5 kilobase region. This region falls between prospero (a transcription factor involved in neurogenesis) and the master sex-determining gene doublesex. It contains the 5′-UTR and cis-regulatory domain of doublesex, and no coding sequence. Wing size reduction correlates with an increase in doublesex expression level that is specific to developing male wings. Our results indicate that non-coding changes are responsible for recent divergence in sex-specific morphology between two closely related species. We have not yet resolved whether wing size evolution at the ws1 locus is caused by regulatory alterations of dsx or prospero, or by another mechanism. This study demonstrates the feasibility of efficient positional cloning of quantitative trait loci (QTL) involved in a broad array of phenotypic differences among Nasonia species.

Attenuation of porcine reproductive and respiratory syndrome virus strain MN184 using chimeric construction with vaccine sequence

Two genetically distinct infectious recombinant virus clones (pMLV, constructed from Ingelvac® PRRS MLV and pMN184, constructed from virulent strain MN184) were developed to study attenuation of contemporary porcine reproductive and respiratory syndrome virus (PRRSV) strain MN184. Two reciprocal chimeric clones (pMLVORF1/MN184 and pMN184ORF1/MLV) were then constructed, such that the 5′UTR/ORF1 of one genotype was linked to ORF2–7/3′UTR from the other genotype. In vitro studies demonstrated that the rescued chimeric viruses possessed intermediate growth properties compared to recombinant rMLV and rMN184. Swine inoculation with rMN184 and rMLV verified that these viruses fully mimicked the respective parent virus. In addition, earlier and higher antibody responses were detected in animals infected with rMN184 in contrast to those infected with rMLV. Chimeric virus treatment groups showed similar antibody responses as seen with these parent viruses, but much less severe pathogenesis when compared to the rMN184 group. These data suggested that genetic aspects of Ingelvac® PRRS MLV 5′UTR/ORF1 replicase region and/or the structural proteins/3′UTR can serve to attenuate virulent strain MN184. The data also indicated that designed PRRSV vaccines could be developed, keeping the known 5′UTR/replicase region of an early vaccine strain such as Ingelvac® PRRS MLV intact, but replacing the structural protein/3′UTR domain with that of an emerging virulent virus.

Characterisation of salmon and trout CD8α and CD8β

The genes and corresponding cDNAs of both α and β chains of the Atlantic salmon ( Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for α and β chains of brown trout ( Salmo trutta) and for the β chain in rainbow trout ( Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5′ UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3′ UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15–20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRα gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8β in a teleost and together with the CD8α sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

Role of the 3'-untranslated region of human endothelin-1 in vascular endothelial cells. Contribution to transcript lability and the cellular heat shock response.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post-transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-1 and luciferase transcripts with or without a wild type ET-1 3'-untranslated region (3'-UTR) and found that the 3'-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3'-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10-nucleotide region at position 978-987 of the 3'-UTR. Depletion of AUF1 by RNA interference up-regulated ET-1 in endothelial cells suggesting AUF1-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3'-UTRs were also stabilized in response to proteasome inhibition whereas DE1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3'-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock.

Mutational analysis of the different bulge regions of hepatitis C virus domain II and their influence on internal ribosome entry site translational ability.

The hepatitis C virus (HCV) 5'-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.

Prominent expression of the selenoprotein thioredoxin reductase in the medullary rays of the rat kidney and thioredoxin reductase mRNA variants differing at the 5′ untranslated region

The mammalian selenoprotein thioredoxin reductase is a central enzyme in protection against oxidative damage or the redox control of cell function. Previously a neuroblastoma-cell-derived 2193 bp cDNA for rat thioredoxin reductase 1 (TrxR1) was characterized [Zhong, Arnér, Ljung, Aslund and Holmgren (1998) J. Biol. Chem. 273, 8581-8591]. Here, the major rat TrxR1 mRNA was determined as 3.5 kb by Northern blotting. A corresponding full-length 3360 bp liver-derived cDNA was cloned and sequenced, being extended in the 3' untranslated region (3' UTR) compared with the previous clone. Among tissues examined, lowest TrxR1 mRNA levels were found in spleen and testis and highest in liver and kidney. High expression in kidney was unexpected and in situ hybridization of adult rat kidney was performed. This revealed a highly structured expression pattern with the mRNA being prominently synthesized in the proximal tubules of the medullary rays. Analysing rat kidney cDNA, a 5' UTR domain of TrxR1 was found that was different from that in liver- or neuroblastoma-derived cDNA clones. The kidney-derived 5' UTR mRNA domain was instead highly similar to kidney-derived cDNA variants of murine apolipoprotein E. By sequence determination of the rat genomic sequence upstream of the open reading frame for TrxR1, an exon was encountered that encoded a third alternative 5' UTR domain that could also be expressed, as confirmed by its presence in a mouse skin TrxR1 cDNA clone. It can therefore be concluded that TrxR1 mRNA is expressed in at least three different variants that differ at their 5' UTRs.

Comparative analysis of two assays for genotyping hepatitis C virus basedon genotype-specific primers or probes

Background/Aims: The importance of determining the hepatitis C virus genotype has increased since several authors have described a good correlation between hepatitis C virus genotype and response to interferon treatment or to disease severity. It is thus of particular importance to develop reliable assays for hepatitis C virus genotyping. Methods: We have comparatively analyzed hepatitis Cvirus genotypes of 208 French and Italian chronically infected patients using genotype-specific primers polymerase chain reaction in the capsid region and a genotype-specific probe-based assay in the 5′ untranslated region (LiPA). Results: We found a good concordance between the two assays for the prevalent genotypes in our regions (145/174). The nucleotide sequences in the 5′UTR and capsid domain were investigated to determine the molecular basis of some discordant results. This analysis showed that the genotype-specific probe based assay gave more consistent results, probably because this technique is based on several probes distributed along the 5′UTR to reveal each genotype. In contrast, the low variability of 5′UTR did not always permit classification of the hepatitis C virus genotype 1 subtypes. We also found some problems, using genotype-specific primers polymerase chain reaction, for less represented genotypes; in particular, in the presence of type 2a/III we obtained more discordant results. This observation is of importance in view of the potential association of this genotype with mild liver disease and a good response to interferon-a treatment.