Untranslated Region Deletion Research Articles

Molecular basis of RNA recombination in the 3'UTR of chikungunya virus genome.

Chikungunya virus (CHIKV) is a rapidly spreading re-emergent virus transmitted from mosquitoes to humans. The emergence of epidemic variants has been associated with changes in the viral genome, such as the duplication of repeated sequences in the 3' untranslated region (UTR). Indeed, blocks of repeated sequences seemingly favor RNA recombination, providing the virus with a unique ability to continuously change the 3'UTR architecture during host switching. In this work, we provide experimental data on the molecular mechanism of RNA recombination and describe specific sequence and structural elements in the viral 3'UTR that favor template switching of the viral RNA-dependent RNA polymerase on the 3'UTR. Furthermore, we found that a 3'UTR deletion mutant that exhibits markedly delayed replication in mosquito cells and impaired transmission in vivo, recombines in reference laboratory strains of mosquitoes. Altogether, our data provide novel experimental evidence indicating that RNA recombination can act as a nucleic acid repair mechanism to add repeated sequences that are associated to high viral fitness in mosquito during chikungunya virus replication.

A 44-Nucleotide Region in the Chikungunya Virus 3' UTR Dictates Viral Fitness in Disparate Host Cells.

We previously reported that deletion of a 44-nucleotide element in the 3' untranslated region (UTR) of the Chikungunya virus (CHIKV) genome enhances the virulence of CHIKV infection in mice. Here, we find that while this 44-nucleotide deletion enhances CHIKV fitness in murine embryonic fibroblasts in a manner independent of the type I interferon response, the same mutation decreases viral fitness in C6/36 mosquito cells. Further, the fitness advantage conferred by the UTR deletion in mammalian cells is maintained in vivo in a mouse model of CHIKV dissemination. Finally, SHAPE-MaP analysis of the CHIKV 3' UTR revealed this 44-nucleotide element forms a distinctive two-stem-loop structure that is ablated in the mutant 3' UTR without altering additional 3' UTR RNA secondary structures.

Statistical analysis supports UTR (untranslated region) deletion theory in SARS-CoV-2

ABSTRACT It was noticed that the mortality rate of SARS-CoV-2 infection experienced a significant declination in the early stage of the epidemic. We suspect that the sharp deterioration of virus toxicity is related to the deletion of the untranslated region (UTR) of the virus genome. It was found that the genome length of SARS-CoV-2 engaged a significant truncation due to UTR deletion after a mega-sequence analysis. Sequence similarity analysis further indicated that short UTR strains originated from its long UTR ancestors after an irreversible deletion. A good correlation was discovered between genome length and mortality, which demonstrated that the deletion of the virus UTR significantly affected the toxicity of the virus. This correlation was further confirmed in a significance analysis of the genetic influence on the clinical outcomes. The viral genome length of hospitalized patients was significantly more extensive than that of asymptomatic patients. In contrast, the viral genome length of asymptomatic was considerably longer than that of ordinary patients with symptoms. A genome-level mutation scanning was performed to systematically evaluate the influence of mutations at each position on virulence. The results indicated that UTR deletion was the primary driving force in alternating virus virulence in the early evolution. In the end, we proposed a mathematical model to explain why this UTR deletion was not continuous.

Functional Investigation of the Gene NFKBIZ and the Impact of 3'UTR Mutations in Diffuse Large B-Cell Lymphoma

Introduction:Diffuse large B-cell lymphoma (DLBCL) represents the most frequent non-Hodgkin lymphoma, accounting for over 30% of cases. The two main subtypes of DLBCL, germinal center B-cell-like (GCB) and activated B-cell-like (ABC), have distinct clinical outcomes, unique gene expression profiles and a growing list of defining genetic features. Recurrent somatic mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to constitutively activate NF-κB signaling, either directly or indirectly, in ABC DLBCL. Amplification of the NFKBIZ locus has been observed in ~10% of ABC DLBCL cases however, no other mutations affecting this locus have been previously described. Although some alterations leading to NF-κB activation have been identified and characterized, there remain ABC cases with NF-κB activation and no clear genetic explanation. Through a meta-analysis of published and in-house exome and genome sequence data for recurrent mutations affecting non-coding regions, we found NFKBIZ had the strongest enrichment for mutations within the 3' untranslated region (UTR). The majority of these mutations affect a highly conserved region of the mRNA that is predicted to form multiple stem-loops or more complex secondary structures (Figure 1A). The endoribonuclease Regnase-1, encoded by the gene ZC3H12A, targets mRNAs with conserved secondary structures in the 3'UTR such as those predicted to form in NFKBIZ . Mutations in the NFKBIZ 3'UTR potentially abrogate this interaction with Regnase-1.Aims and Methods:Based on the observed mutation pattern, we hypothesized that NFKBIZ 3'UTR mutations are more significantly enriched in ABC DLBCL and lead to increased activation of the NF-κB signaling pathway. We aimed to (1) determine the frequencies of mutations in these two genes in DLBCL, specifically within each sub-type in our cohort and (2) investigate the effects of NFKBIZ 3'UTR mutations on mRNA and protein expression in activating the NF-κB signaling pathway in B-cells. We analyzed RNA-seq data and performed targeted sequencing on these lines and 334 primary DLBCL tumors and 8 DLBCL cell lines. We assessed mRNA abundance and the allelic abundance of mutant and wild-type NFKBIZ using RNA-seq reads and a custom “wild-type negative” or “drop-off” droplet digital PCR (ddPCR) assay. The selected cell lines represent those naturally NFKBIZ mutant (amplification or containing a 3'UTR deletion), MYD88 mutant, or wild-type for both. These lines were used to investigate NFKBIZ mRNA and IκB-ζ protein expression.Results:Targeted sequencing and copy number analysis of 316 cases with available RNA-seq data uncovered 66 DLBCLs (21% of cases) containing any NFKBIZ mutation, 34 (10.8%) of which were 3'UTR mutations, and 19 DLBCLs with a mutation in ZC3H12A (6% of cases) . A substantial fraction of the NFKBIZ mutations were confirmed somatic by sequencing matched constitutional DNA. NFKBIZ 3'UTR mutations were mutually exclusive with MYD88 mutations and largely segregated from those affecting ZC3H12A (Figure 1B). In aggregate, mutations in either of these novel genes are present in 22.6% of all DLBCLs and 30.8% of ABC cases. The enrichment for these mutations in ABC cases was highly significant (OR 4.1, 95% CI 2.04-8.48). Results from our ddPCR experiments revealed allelic imbalance of NFKBIZ mRNA in mutant cell lines, specifically a relative increase of the mutant transcript. Patient RNA samples harboring NFKBIZ mutations (small deletions and SNVs) showed similar results. Western blot experiments confirmed increased levels of IκB-ζ protein consistent with increased mRNA in all NFKBIZ and MYD88 mutant cell lines (Figure 1C).Conclusion:Our current work has shown promising results indicating that NFKBIZ may be an underappreciated driver gene in ABC DLBCL. We have demonstrated that mutations affecting the 3'UTR of the NFKBIZ gene are significantly enriched in the ABC sub-type. These mutations cause deregulation of the mRNA and lead to over-expression of the IκB-ζ protein which activates the NF-κB signaling pathway. Uncovering new ways in which the NF-κB pathway can be dysregulated in ABC DLBCL will allow for better treatments to be developed and more accurate stratification of patients into the clinical trials that will be the most beneficial to their specific set of mutations. [Display omitted] DisclosuresConnors:Bristol-Myers Squibb: Research Funding; Genentech: Research Funding; Merck: Research Funding; Lilly: Research Funding; Cephalon: Research Funding; F Hoffmann-La Roche: Research Funding; Janssen: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Amgen: Research Funding; NanoString Technologies: Research Funding. Scott:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; BCCA: Patents & Royalties: Patent describing molecular subtyping of DLBCL licensed to NanoString Technologies. Patent describing measurement of the proliferation signature in MCL.. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Morin:Epizyme, Inc: Consultancy.

Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Chikungunya virus evolution following a large 3'UTR deletion results in host-specific molecular changes in protein-coding regions.

The 3′untranslated region (UTR) in alphavirus genomes functions in virus replication and plays a role in determining virus host range. However, the molecular evolution of virus UTRs is understudied compared to the evolution of protein-coding regions. Chikungunya virus (CHIKV) has the longest 3′UTR among the alphaviruses (500–700 nt), and 3′UTR length and sequence structure vary substantially among different CHIKV lineages. Previous studies showed that genomic deletions and insertions are key drivers of CHIKV 3′UTR evolution. Inspired by hypothesized deletion events in the evolutionary history of CHIKV, we used experimental evolution to examine CHIKV adaptation in response to a large 3′UTR deletion. We engineered a CHIKV mutant with a 258 nt deletion in the 3′UTR (ΔDR1/2). This deletion reduced viral replication on mosquito cells, but did not reduce replication on mammalian cells. To examine how selective pressures from vertebrate and invertebrate hosts shape CHIKV evolution after a deletion in the 3′UTR, we passaged ΔDR1/2 virus populations strictly on primate cells, strictly on mosquito cells, or with alternating primate/mosquito cell passages. We found that virus populations passaged on a single host cell line increased in fitness relative to the ancestral deletion mutant on their selected host, and viruses that were alternately passaged improved on both hosts. Surprisingly, whole genome sequencing revealed few changes in the 3′UTR of passaged populations. Rather, virus populations evolved improved fitness through mutations in protein coding regions that were associated with specific hosts.

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

The Role of mRNA Degradation in Dynamic Nitrogen Environments in Saccharomyces cerevisiae

Studying the reprogramming of gene expression is fundamental to understanding cellular growth, development and stem cell differentiation. In Saccharomyces cerevisiae cells undergo reprogramming of gene expression when they are grown in nitrogen‐poor conditions and then switched to nitrogen‐rich conditions. This is controlled by the nitrogen catabolite repression (NCR) response. In a previous study, NCR transcripts were shown to undergo accelerated degradation when the cells where switched from growth on proline in a nitrogen‐poor environment to growth in a nitrogen‐rich environment upon the addition of glutamine. This study aims to determine the benefit of accelerated mRNA degradation in dynamic nitrogen environments by studying the effects of mutants of NCR repressors during this transition. We suspect that knocking out the repressors will stop the accelerated degradation once glutamine is added. This will in turn affect the growth rates and cause the mutants to have longer lag phase, a period where there is no detectable change in the growth. The general amino acid permease (GAP1) was used in representation of NCR transcripts to study the effects. This gene encodes a permease that senses the presence of amino acids substrates to regulate localization to the plasma membrane when needed. We identified mutants where GAP1 is stabilized in a nitrogen‐rich environment by using knock‐outs (KO) of the NCR repressor genes Ure2, Dal80 and Gzf3. The Ure2KO mutant showed depression of GAP1 in nitrogen‐rich conditions, but it caused a growth defect when the cells were grown in nitrogen‐poor conditions. In addition, we tested the effect of the GAP1 5′ untranslated region (UTR) mutant on growth dynamics because it has been shown to stabilize the GAP1 transcript during the transition from nitrogen‐poor environment to a nitrogen‐rich environment. A T‐test was performed to see if there was any difference between the growth rates of the wildtype and the 5′UTR deletion. Turns out that there was no significant difference between them before (p value=0.13) and after the glutamine addition (p value= 0.15), despite there being a notable distinction between the slopes. When the graphs for the nitrogen up shift were generated a lag phase was detected and it appeared to be an hour longer in the mutant. Which indicates that accelerated degradation is an important process in dynamic nitrogen environments.Support or Funding InformationNIH‐MARC Program undergraduate fellowship (5T34GM00781‐37)

An Indel Polymorphism in the MtnA 3' Untranslated Region Is Associated with Gene Expression Variation and Local Adaptation in Drosophila melanogaster.

Insertions and deletions (indels) are a major source of genetic variation within species and may result in functional changes to coding or regulatory sequences. In this study we report that an indel polymorphism in the 3’ untranslated region (UTR) of the metallothionein gene MtnA is associated with gene expression variation in natural populations of Drosophila melanogaster. A derived allele of MtnA with a 49-bp deletion in the 3' UTR segregates at high frequency in populations outside of sub-Saharan Africa. The frequency of the deletion increases with latitude across multiple continents and approaches 100% in northern Europe. Flies with the deletion have more than 4-fold higher MtnA expression than flies with the ancestral sequence. Using reporter gene constructs in transgenic flies, we show that the 3' UTR deletion significantly contributes to the observed expression difference. Population genetic analyses uncovered signatures of a selective sweep in the MtnA region within populations from northern Europe. We also find that the 3’ UTR deletion is associated with increased oxidative stress tolerance. These results suggest that the 3' UTR deletion has been a target of selection for its ability to confer increased levels of MtnA expression in northern European populations, likely due to a local adaptive advantage of increased oxidative stress tolerance.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3'UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3'UTR that may act as regulatory sequences. Analysis of ODC 3'UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

β(2)-adrenergic receptors (β(2)-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β(2)-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β(2)-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β(2)-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β(2)-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β(2)-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.

Post-transcriptional Regulation of Neuro-oncological Ventral Antigen 1 by the Neuronal RNA-binding Proteins ELAV

Alternative splicing of pre-mRNAs plays an important role in generating biological and functional diversity. Neuro-oncological ventral antigen 1 (Nova1) is a neuron-specific splicing factor that controls the alternative processing of a wide array of mRNAs important for synaptic activity. It is essential for the proper development of the mammalian motor system and for the survival of motoneurons. Because Nova1 gene contains putative regulatory AU-rich elements (ARE) in its highly conserved 3'-untranslated region, we investigated whether its expression is regulated by post-transcriptional mechanisms mediated by ARE-binding proteins. Among these, the neuronal ELAV (nELAV) factors are interesting candidates, because their RNA binding activity is necessary for neuronal differentiation and maintenance. By analysis of ribonucleoprotein complexes in vivo and in vitro we demonstrated that the Nova1 mRNA is a novel target of the nELAV proteins. We defined the nELAV binding site by functional experiments with luciferase reporter gene and Nova1 3'-untranslated region deletion sequences. Gene silencing and overexpression of the nELAV member HuD in motoneuronal NSC34 cells indicate that Nova1 mRNA stability and translation are positively and strongly controlled by the nELAV proteins. In addition, nELAV phosphorylation by a PKC-dependent pathway induces the recruitment of Nova1 mRNA to polysomes. Noteworthy, we found that nELAV proteins are also able to modulate Nova1 splicing activity on its target genes. Our data indicate nELAV proteins as the first factors affecting the expression and activity of the neuronal splicing regulator Nova1 and, consequently, as major candidates for the physiological modulation of Nova1-dependent processing of pre-mRNAs in neurons.

Variation in the miRNA-433 Binding Site of FGF20 Confers Risk for Parkinson Disease by Overexpression of α-Synuclein

Parkinson disease (PD) is a common neurodegenerative disorder caused by environmental and genetic factors. We have previously shown linkage of PD to chromosome 8p. Subsequently, fibroblast growth factor 20 (FGF20) at 8p21.3-22 was identified as a risk factor in several association studies. To identify the risk-conferring polymorphism in FGF20, we performed genetic and functional analysis of single-nucleotide polymorphisms within the gene. In a sample of 729 nuclear families with 1089 affected and 1165 unaffected individuals, the strongest evidence of association came from rs12720208 in the 3' untranslated region of FGF20. We show in several functional assays that the risk allele for rs12720208 disrupts a binding site for microRNA-433, increasing translation of FGF20 in vitro and in vivo. In a cell-based system and in PD brains, this increase in translation of FGF20 is correlated with increased alpha-synuclein expression, which has previously been shown to cause PD through both overexpression and point mutations. We suggest a novel mechanism of action for PD risk in which the modulation of the susceptibility gene's translation by common variations interfere with the regulation mechanisms of microRNA. We propose this is likely to be a common mechanism of genetic modulation of individual susceptibility to complex disease.

Prognostic role of thymidylate synthase (TS) gene polymorphisms in gastric cancer patients treated with surgery and adjuvant chemotherapy

4044 Background: Previous studies suggest that TS expression in tumor tissue is related with both sensitivity to 5-FU-based chemotherapy and patients’outcome. TS gene has polymorphic sequences in its untranslated region (UTR) which are associated with TS expression. Aim of this study is to evaluate prognostic role of the TS polymorphisms in gastric cancer patients treated with surgery and 5-FU-based adjuvant chemotherapy. Methods: Ninety patients with advanced gastric cancer who had undergone radical surgery and received 5-FU-based adjuvant chemotherapy were included in this study. All patients underwent a 44-months minimum follow-up time. Lymphocyte genomic DNA was extracted from peripheral blood samples of the patients. Genotypes of variable number of tandem repeat (VNTR) and G/C single nucleotide polymorphism (SNP) in the TS 5’-UTR and 6bp deletion polymorphism in the 3’-UTR were determined by PCR and PCR-RFLP. According to previous studies the VNTR and the SNP were combined and the genotypes were grouped as follows; 5’-high (3G/3G, 3G/3C, 2R/3G) and 5’-low (2R/2R, 2R/3C, 3C/3C). Genotypes in 3’-UTR were also classified as follows; 3’-high (ins6/ins6) and 3’-low (del6/ins6, del6/del6). Results: There were no significant relationships between clinico-pathologic features and each TS polymorphism. Ten patients were 5’-high/3’-high (100% relapse/death), 36 patients were 5’-high/3’-low (67% relapse/death, 33% disease-free), 19 patients were 5’-low/3’-high (58% relapse/death, 42% disease-free), 25 patients were 5’-low/3’-low (36% relapse/death, 64% disease-free). Patients with 5’-low/3’-low genotypes showed significantly better outcome than those with 5’-high/3’-low and 5’-high/3’-high genotypes in regard to both 3-year disease-free and overall survival rates. The presence of at least one high TS expression genotype showed independent adverse prognostic role in multivariate analysis. Conclusions: TS polymorphisms showed prognostic influence in the studied population of gastric cancer patients. Analysis of combined TS5’- and TS3’-UTR genotypes may be useful for improving adjuvant treatment strategies with tailored chemotherapy. No significant financial relationships to disclose.

Risk factors for neural tube defects: associations between uncoupling protein 2 polymorphisms and spina bifida.

Polymorphisms in the mitochondrial membrane transporter gene UCP2 are capable of affecting energy metabolism, body weight regulation, and possibly preventing the buildup of reactive oxygen species, all factors that could contribute to neural tube defect risk through maternal obesity and diabetes. Genomic DNA was extracted from newborn screening blood spots obtained from infants with spina bifida and nonmalformed control infants. Genotype frequencies of two genetic variants in the UCP2 gene, an amino acid substitution of valine for alanine at codon 55 in exon 4, and a 45-base pair insertion/deletion in the 3' untranslated region of exon 8,were determined by restriction enzyme digestion of PCR amplification products. We found the frequency of the 3' untranslated region deletion homozygous genotype (256/256) as well as the A55V homozygous (Val/Val) genotype to be higher in SB infants than in controls (odds ratio [OR], 3.1; 95% confidence interval [CI], 0.9-10.4 and OR = 2.0; 95% CI = 0.3-11.1, respectively). Additionally, the frequency of the combined homozygous 256/256,+ / + genotype was higher in cases and resulted in more than a threefold higher spina bifida risk (OR = 3.6; 95% CI = 1.0-13.1). These data are the first to suggest that polymorphisms in the UCP2 gene may be genetic risk factors of spina bifida.

Inversions in the Chlamydomonas chloroplast genome suppress a petD 5' untranslated region deletion by creating functional chimeric mRNAs.

FUD6 is a non-photosynthetic Chlamydomonas mutant that lacks the cytochrome b6/f complex, due to a 236 bp deletion that removes the promoter and part of the 5' untranslated region (UTR) of the chloroplast petD gene, which encodes subunit IV of the complex. Two photosynthetic revertants of FUD6 that synthesized wild-type levels of subunit IV were found to contain related inversions of the chloroplast genome that resulted from recombination between small inverted repeats. These inversions created a functional chimeric petD gene that includes the promoter and part of the 5' UTR of the newly identified ycf9-psbM transciption unit, fused to the petD 5' UTR upstream of the FUD6 deletion. Accumulation of the ycf9-psbM dicistronic transcript was disrupted in the revertants, but monocistronic psbM mRNA accumulated normally. The FUD6 revertants demonstrate the ability of the Chlamydomonas chloroplast genome to undergo a large inversion without a deleterious effect on chloroplast function, reminiscent of events that have led to the evolutionary divergence of chloroplast genomes.

The 3' untranslated region of IL-1beta regulates protein production.

IL-1beta, a pro-inflammatory cytokine, has sequences in its 3' untranslated region (UTR) that may play a role in the post-transcriptional regulation of IL-1beta production. To test this hypothesis, a series of chimeric reporter genes were developed consisting of a chloramphenicol acetyltransferase (CAT) gene the native 3'-UTR of which was replaced by the full-length IL-1beta 3'-UTR or various 3'-UTR deletion mutants. Expression of these constructs under the SV40 late promoter in THP-1 cells showed that the full-length 3'-UTR repressed constitutive CAT activity to 28% of control CAT activity. Further analysis of the 3'-UTR localized the repressor signal to an adenosine-thymidine (AdT)-rich region. Upon exposure to LPS, the full-length IL-1beta 3'-UTR mediated almost a fivefold increase in CAT activity. The LPS response was not simply loss of AdT-mediated repression; when this sequence was tested alone, it did not respond to LPS. The LPS response effect was localized to the terminal 177 base pairs of the IL-1beta 3'-UTR. The increase in CAT activity following LPS stimulation was associated with an increased CAT.IL-1beta 3'-UTR mRNA half-life, indicating at least one effect of the 3'-UTR on a post-transcriptional process. These studies demonstrate that the IL-1beta 3'-UTR is involved in the regulation of IL-1beta protein production, and a LPS response element may be in the IL-1beta 3'-UTR.

Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mamalian cells.

The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investigated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by deleting either the 5' untranslated region (UTR), the 3'UTR, or both UTRs. The 5'UTR contains two false AUG initiation codons followed by a stop codon. The cDNAs in the eukaryotic expression vector pSV2-NEO-ECE, as well as the 5'3'UTR-truncated cDNA in pSVL, were stably transfected into Chinese hamster ovary cells. Pools of more than 100 colonies were studied in order to minimize insertion site-dependent variation in the level of expression. Scatchard analysis of TSH binding indicated that the number of receptors on the surface of Chinese hamster ovary cells expressing the wild-type transcript (approximately 16,000/cell) increased approximately 2-fold with 5'UTR deletion, approximately 5-fold with 3'UTR deletion, and approximately 10-fold with both 5'UTR and 3'UTR deletion. TSH binding affinities of all constructs were in the range of 2-5 x 10(-10) M. No significant difference was evident between the 5'3'UTR truncated cDNAs in the two different vectors, pSV2-NEO-ECE and pSVL. The increase in the amplitude of the cAMP response to TSH stimulation was commensurate with the number of receptors expressed on the surface of the different cell lines. Truncation of the 5'UTR did not alter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as well as the 5'3'UTR-deleted transcript, increased approximately 4-fold independent of the expression vector used. In summary, both the 5'UTR and 3'UTR of the human TSH receptor mRNA influence the level of receptor expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the MRNA. These data explain the greater level of TSH receptor expression in cell lines that are transfected with cDNA lacking these regions of the mRNA transcript.