scholarly journals Functional studies of the 5′-untranslated region of human 5-HT4 receptor mRNA

2005 ◽  
Vol 387 (2) ◽  
pp. 463-471 ◽  
Author(s):  
Marjorie MAILLET ◽  
Monique GASTINEAU ◽  
Pascal BOCHET ◽  
Marie-Liesse ASSELIN-LABAT ◽  
Eric MOREL ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Mutational Analysis ◽  
Receptor Gene ◽  
Luciferase Reporter ◽  
Regulatory Sequence ◽  
Cellular Functions ◽  
Luciferase Reporter Gene ◽  
Enhancer Activity ◽  
Rnase Protection ◽  
Functional Studies

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5′-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5′-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase–PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at −3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (−3299/−3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (−220/−61) located in the long 5′-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.

scholarly journals Cap-Independent Translational Enhancement by the 3′ Untranslated Region of Red Clover Necrotic Mosaic Virus RNA1

2003 ◽  
Vol 77 (22) ◽  
pp. 12113-12121 ◽  
Author(s):  
Hiroyuki Mizumoto ◽  
Masahiro Tatsuta ◽  
Masanori Kaido ◽  
Kazuyuki Mise ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Untranslated Region ◽  
Red Clover ◽  
Luciferase Reporter ◽  
Base Substitution ◽  
Luciferase Reporter Gene ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Translational Activity ◽  
Independent Translation

ABSTRACT Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3′ ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5′ cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5′ cap and 3′ poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5′ cap- and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3′ untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5′ cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3′ UTR, designated the 3′ translation element of Dianthovirus RNA1 (3′TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and Luteovirus. A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3′TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.

scholarly journals A distal activation domain is critical in the regulation of the rat androgen receptor gene promoter

1993 ◽  
Vol 294 (3) ◽  
pp. 779-784 ◽  
Author(s):  
C S Song ◽  
S Her ◽  
M Slomczynska ◽  
S J Choi ◽  
M H Jung ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Nucleotide Sequence ◽  
Receptor Gene ◽  
Critical Role ◽  
Androgen Receptor Gene ◽  
Luciferase Reporter ◽  
Upstream Region ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Cis Element

The far upstream region of the rat androgen receptor (AR) gene has been cloned, and the nucleotide sequence up to -2656 bp established. Nested deletion mutants of rat AR 5′ flanking sequences were ligated to the luciferase reporter gene, and their promoter activities were examined in transfected COS1 cells. Results show a critical cis-acting domain located between positions -960 and -940. Deletion of this cis element resulted in a greater than 90% decrease in the promoter activity. A nuclear protein that specifically binds to this 21-nucleotide sequence was identified by gel mobility shift analysis. The -960/-940 cis element has no identify to the binding sequence of any known transcription factor. Furthermore, the cognate binding protein is present in both rat and human (HeLa) cell nuclear extracts. We conclude that a novel trans-activator interacting at the -960/-940 region plays a critical role in the regulation of AR gene expression.

scholarly journals miR-373-3p inhibits EMT via regulation of TGFβR2 in choriocarcinoma

2019 ◽  
Author(s):  
yanjie lu ◽  
Xiaoru Li ◽  
Yanzhen Zuo ◽  
qian xu ◽  
lei liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Untranslated Region ◽  
Bioinformatic Analysis ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Luciferase Reporter Gene ◽  
Early Metastasis ◽  
Choriocarcinoma Cells ◽  
Jar Cells

Abstract Background: Previous studies have indicated that early metastasis is a major cause of mortality in patients with choriocarcinoma. However, what determines whether early metastasis of choriocarcinoma has occurred is unknown. The emerging role of miRNA in regulating cancer development and progression has been recognized. MiR-373-3p has been shown to play pivotal roles in tumorigenesis and metastasis. However, whether miR-373-3p functions to promote choriocarcinoma metastasis is not clear. The purpose of this study is to determine the function of miR-373-3p in the progression of choriocarcinoma. Methods: In this study, we first compared EMT-related markers, which are inversely correlated with miR-373-3p expression, in trophoblast and choriocarcinoma cell lines. Using PCR and western blot, the upregulation of miR‑373‑3p was observed to inhibit EMT progression. Similarly, gain-and loss-of-function studies revealed that ectopic miR-373-3p overexpression inhibited the metastasis of choriocarcinoma cells. Results: Our results revealed that miR-373-3p functions as an inhibitor in JEG-3 and JAR cells; this is due to its mediation of the TGF-β signalling pathway, which is responsible for EMT. The bioinformatic analysis and dual‑luciferase reporter gene assays were employed to verify that miR‑373‑3p might interact with the 3' untranslated region of TGFβR2 mRNA. Further western blot results showed miR‑373‑3 preversed the increases of TGFβR2 and inhibited EMT. Conclusions: In light of our observations, miR‑373‑3p upregulation partly accounts for TGFβR2 downregulation and leads to a restraint of EMT and metastasis. MiR‑373‑3p may, therefore, serve as a valuable target in potential anticancer strategies to treat choriocarcinoma.

scholarly journals MSH2 Overexpression Due to an Unclassified Variant in 3’-Untranslated Region in a Patient with Colon Cancer

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 167
Author(s):  
Raffaella Liccardo ◽  
Antonio Nolano ◽  
Matilde Lambiase ◽  
Carlo Della Ragione ◽  
Marina De Rosa ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Tumor Development ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Unclassified Variant ◽  
Dna Mismatch ◽  
Functional Assays ◽  
Mmr Genes ◽  
Uncertain Significance ◽  
Low Expression

Background: The loss or low expression of DNA mismatch repair (MMR) genes can result in genomic instability and tumorigenesis. One such gene, MSH2, is mutated or rearranged in Lynch syndrome (LS), which is characterized by a high risk of tumor development, including colorectal cancer. However, many variants identified in this gene are often defined as variants of uncertain significance (VUS). In this study, we selected a variant in the 3′ untranslated region (UTR) of MSH2 (c*226A > G), identified in three affected members of a LS family and already reported in the literature as a VUS. Methods: The effect of this variant on the activity of the MMR complex was examined using a set of functional assays to evaluate MSH2 expression. Results: We found MSH2 was overexpressed compared to healthy controls, as determined by RTqPCR and Western blot analyses of total RNA and proteins, respectively, extracted from peripheral blood samples. These results were confirmed by luciferase reporter gene assays. Conclusions: We therefore speculated that, in addition to canonical inactivation via a gene mutation, MMR activity may also be modulated by changes in MMR gene expression.

Regulation of fibrinogen production by microRNAs

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2608-2615 ◽  
Author(s):  
Alexandre Fort ◽  
Christelle Borel ◽  
Eugenia Migliavacca ◽  
Stylianos E. Antonarakis ◽  
Richard J. Fish ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Mirna Precursor ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Quantitative Measurements ◽  
Increased Risk ◽  
Huh7 Cells ◽  
Steady State Levels ◽  
Precursor Molecules

Abstract Elevated levels of fibrinogen are associated with increased risk of cardiovascular disease, whereas low fibrinogen can lead to a bleeding disorder. We investigated whether microRNAs (miRNAs), known to act as post-transcriptional regulators of gene expression, regulate fibrinogen production. Using transfection of a library of 470 annotated human miRNA precursor molecules in HuH7 hepatoma cells and quantitative measurements of fibrinogen production, we identified 23 miRNAs with down-regulating (up to 64% decrease) and 4 with up-regulating effects (up to 129% increase) on fibrinogen production. Among the down-regulating miRNAs, we investigated the mechanism of action of 3 hsa-miR-29 family members and hsa-miR-409-3p. Overexpression of hsa-miR-29 members led to decreased steady-state levels of all fibrinogen gene (FGA, FGB, and FGG) transcripts in HuH7 cells. Luciferase reporter gene assays demonstrated that this was independent of miRNA-fibrinogen 3′-untranslated region interactions. In contrast, overexpression of hsa-miR-409-3p specifically lowered fibrinogen Bβ mRNA levels, and this effect was dependent on a target site in the fibrinogen Bβ mRNA 3′-untranslated region. This study adds to the known mechanisms that control fibrinogen production, points toward a potential cause of variable circulating fibrinogen levels, and demonstrates that a screening approach can identify miRNAs that regulate clinically important proteins.

The function of the bcl-x promoter in erythroid progenitor cells

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2235-2242 ◽  
Author(s):  
Cuixia Tian ◽  
Paul Gregoli ◽  
Maurice Bondurant
Keyword(s):  
Progenitor Cells ◽  
Specific Binding ◽  
Luciferase Reporter ◽  
Erythroid Cell ◽  
Luciferase Reporter Gene ◽  
Sv40 Promoter ◽  
Enhancer Activity ◽  
Sequence Element ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

The protein Bcl-xL is essential for survival of erythroid progenitor cells, and it increases substantially during late erythrocyte differentiation due to an increase of mRNA. We mapped the transcription start sites of bcl-x mRNA in mouse and human erythroblasts, and we analyzed the function of the mousebcl-x promoter by transient and stable transfection assays in a mouse erythroid cell line using plasmids containing thebcl-x promoter fused to a luciferase reporter gene. In mouse erythroblasts, a cluster of start sites at positions −664, −655, and −644 relative to the ATG initiation codon account for almost all transcripts. Human erythroblasts exhibit a start site at −654 that is homologous to the triplet in the mouse. A short sequence element in the mouse bcl-x promoter that includes nucleotides −1804 through −1734 was identified as very important for transcription. This element also showed strong enhancerlike activity in concert with the SV40 promoter in an enhancer test vector. Analyses of mutations indicated that 2 short sequences within the element, about 15 base pair apart, are necessary for full enhancer activity. Gel shift experiments with oligonucleotides representing these sequences revealed specific binding of nuclear proteins from erythroblasts. Some of these proteins are regulated during the late erythroid differentiation.

scholarly journals Structure of the human histamine H1 receptor gene

1998 ◽  
Vol 335 (3) ◽  
pp. 663-670 ◽  
Author(s):  
Marianne D. DE BACKER ◽  
Inge LOONEN ◽  
Peter VERHASSELT ◽  
Jean-Marc NEEFS ◽  
Walter H. M.L. LUYTEN
Keyword(s):  
G Protein ◽  
Untranslated Region ◽  
Receptor Gene ◽  
Promoter Sequence ◽  
Start Codon ◽  
Luciferase Reporter ◽  
H1 Receptor ◽  
Histamine H1 Receptor ◽  
G Protein Coupled ◽  
Human Histamine

Histamine H1 receptor expression has been reported to change in disorders such as allergic rhinitis, autoimmune myocarditis, rheumatoid arthritis and atherosclerosis. Here we report the isolation and characterization of genomic clones containing the 5´ flanking (regulatory) region of the human histamine H1 receptor gene. An intron of approx. 5.8 kb was identified in the 5´ untranslated region, which suggests that an entire subfamily of G-protein-coupled receptors may contain an intron immediately upstream of the start codon. The transcription initiation site was mapped by 5´ rapid amplification of cDNA ends to a region 6.2 kb upstream of the start codon. Immediately upstream of the transcription start site a fragment of 1.85 kb was identified that showed promoter activity when placed upstream of a luciferase reporter gene and transiently transfected into cells expressing the histamine H1 receptor. The promoter sequence shares a number of characteristics with the promoter sequences of other G-protein-coupled receptor encoding genes, including binding sites for several transcription factors, and the absence of TATA and CAAT sequences at the appropriate locations. The promoter sequence described here differs from that reported previously [Fukui, Fujimoto, Mizuguchi, Sakamoto, Horio, Takai, Yamada and Ito (1994) Biochem. Biophys. Res. Commun. 201, 894–901] because the reported genomic clone was chimaeric. Furthermore our study provides evidence that the 3´ untranslated region of the H1 receptor mRNA is much longer than previously accepted. Together, these findings provide a complete view of the structure of the human histamine H1 receptor gene. Both the coding region of the H1 receptor gene and its promoter region were independently mapped to chromosome 3p25.

Functional pleiotropy of an intramolecular triplex-forming fragment from the 3′-UTR of the ratPigrgene

2001 ◽  
Vol 5 (2) ◽  
pp. 53-65 ◽  
Author(s):  
ISABEL FABREGAT ◽  
KATHERINE S. KOCH ◽  
TAKASHI AOKI ◽  
ALLAN E. ATKINSON ◽  
HUONG DANG ◽  
...  
Keyword(s):  
Gene Expression ◽  
Receptor Gene ◽  
Dna Supercoiling ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Luciferase Reporter Gene ◽  
Immunoglobulin Receptor ◽  
293 Cells ◽  
Free Translation ◽  
Luciferase Mrna

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3′-untranslated region (3′-UTR) and inserted into 3′-UTR or 3′ flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5′ flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5′ and 3′ flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3′-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

scholarly journals Cloning and analysis of the CD18 promoter

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2598-2604 ◽  
Author(s):  
AG Rosmarin ◽  
R Levy ◽  
DG Tenen
Keyword(s):  
Messenger Rna ◽  
Transcriptional Start Site ◽  
Cell Surface Expression ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Start Site ◽  
Monocytic Differentiation ◽  
Luciferase Reporter Gene ◽  
Rnase Protection ◽  
Promoter Fragment

Abstract CD18, the common beta chain of the leukocyte integrin adhesion proteins, is expressed exclusively by myeloid cells and lymphocytes. During myeloid differentiation, the increase in CD18 cell surface expression is paralleled by increased CD18 messenger RNA levels. Nuclear run-on studies show that CD18 expression is transcriptionally regulated during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL- 60 monocytic differentiation. The CD18 transcriptional start site was defined by primer extension and RNAse protection. The gene encoding CD18 was cloned and a fragment that overlaps the transcriptional start site was isolated. DNA sequence analysis of this promoter fragment identified potential AP-1 elements that may mediate the TPA transcriptional response. The CD18 promoter also contains a putative binding site for PU.1, a leukocyte-specific transcription factor. DNA elements resembling those found in other myeloid and integrin promoters were identified. The CD18 promoter fragment was linked to the luciferase reporter gene, electroporated into the U937 monocytic cell line, and its expression increased after exposure to TPA. Thus, CD18 may serve as a model for identifying the cis elements and trans-acting factors that regulate gene expression during myeloid differentiation.

scholarly journals A polymorphism in the 3′-untranslated region of the matrix metallopeptidase 9 gene is associated with susceptibility to idiopathic calcium nephrolithiasis in the Chinese population

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052098021
Author(s):  
Qiang Bu ◽  
Yu Zhu ◽  
Qiao-yun Chen ◽  
Hao Li ◽  
Yan Pan
Keyword(s):  
Untranslated Region ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Nucleotide Polymorphisms ◽  
Luciferase Reporter Gene ◽  
Chinese Han ◽  
Gene Assay ◽  
The Matrix ◽  
Matrix Metallopeptidase ◽  
Matrix Metallopeptidase 9

Objective To investigate whether single nucleotide polymorphisms (SNPs) in the 3′ untranslated region (UTR) of the matrix metallopeptidase 9 gene ( MMP9) are associated with susceptibility to calcium oxalate stones. Methods A total of 428 patients with kidney stone disease (KSD) and 450 control individuals were enrolled. Three MMP9 SNPs (rs20544, rs9509, and rs1056628) were genotyped, and MMP9 mRNA and protein expression was determined in patients and controls. The dual luciferase reporter gene assay was conducted by transfecting HEK293 cells with miR-491-5p mimics and plasmids containing MMP9 with rs1056628 AA/CC genotypes. Results The rs1056628 CC genotype was significantly increased in KSD patients compared with controls (CC vs AA: odds ratio [OR] = 2.279, 95% confidence interval [CI] = 1.048–4.956). The rs1056628 C allele frequency was higher in KSD patients than controls. The increased KSD risks associated with rs1056628 were more evident in individuals aged <30 years (OR = 3.504, 95% CI = 1.102–11.139) and men (OR = 2.522, 95% CI = 1.004–6.334). mRNA and protein levels of MMP9 were significantly higher in KSD patients with the CC genotype than in those with the AA genotype. Conclusion This study demonstrates that MMP9 SNP rs1056628 is associated with a significant KSD risk in Chinese Han individuals.

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