scholarly journals Translational efficiency is regulated by the length of the 3' untranslated region.

1996 ◽  
Vol 16 (1) ◽  
pp. 146-156 ◽  
Author(s):  
R L Tanguay ◽  
D R Gallie
Keyword(s):  
Cho Cells ◽  
Posttranscriptional Regulation ◽  
Untranslated Region ◽  
Stop Codon ◽  
Chinese Hamster ◽  
Firefly Luciferase ◽  
Translational Efficiency ◽  
Coding Region ◽  
The Stability ◽  
Reporter Mrna

All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on translational efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, translational efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the translational efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the translational efficiency and the stability of an mRNA.

scholarly journals Regulation of Translational Efficiency by Disparate5′-UTRs of PPARγSplice Variants

PPAR Research ◽  
2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Shawn McClelland ◽  
Roopali Shrivastava ◽  
Jheem D. Medh
Keyword(s):  
Secondary Structure ◽  
Posttranscriptional Regulation ◽  
Protein A ◽  
Firefly Luciferase ◽  
Translational Efficiency ◽  
Protein Levels ◽  
Gene Assay ◽  
Firefly Luciferase Gene

The PPAR-γgene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the5′-untranslated region (UTR). The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the5′-UTR in regulating the efficiency with which the message is translated to protein. A coupledin vitrotranscription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. Anin vivoreporter gene assay using each5′-UTR upstream of the firefly luciferase gene showed that the5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the5′-UTR, and the length of the5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

scholarly journals Regulation of protein synthesis by eIF4E phosphorylation in adult cardiocytes: the consequence of secondary structure in the 5′-untranslated region of mRNA

2004 ◽  
Vol 378 (1) ◽  
pp. 73-82 ◽  
Author(s):  
William J. TUXWORTH ◽  
Atif N. SAGHIR ◽  
Laura S. SPRUILL ◽  
Donald R. MENICK ◽  
Paul J. McDERMOTT
Keyword(s):  
Protein Synthesis ◽  
Secondary Structure ◽  
Untranslated Region ◽  
Primary Cultures ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Hypertrophic Growth ◽  
Reporter Mrna

In adult cardiocytes, eIF4E (eukaryotic initiation factor 4E) activity and protein synthesis are increased concomitantly in response to stimuli that induce hypertrophic growth. We tested the hypothesis that increases in eIF4E activity selectively improve the translational efficiency of mRNAs that have an excessive amount of secondary structure in the 5´-UTR (5´-untranslated region). The activity of eIF4E was modified in primary cultures of adult cardiocytes using adenoviral gene transfer to increase either the amount of eIF4E or the extent of endogenous eIF4E phosphorylation. Subsequently, the effects of eIF4E on translational efficiency were assayed following adenoviral-mediated expression of luciferase reporter mRNAs that were either ‘stronger’ (less structure in the 5´-UTR) or ‘weaker’ (more structure in the 5´-UTR) with respect to translational efficiency. The insertion of G+C-rich repeats into the 5´-UTR doubled the predicted amount of secondary structure and was sufficient to reduce translational efficiency of the reporter mRNA by 48±13%. Translational efficiency of the weaker reporter mRNA was not significantly improved by overexpression of wild-type eIF4E when compared with the stronger reporter mRNA. In contrast, overexpression of the eIF4E kinase Mnk1 [MAP (mitogen-activated protein) kinase signal-integrating kinase 1] was sufficient to increase the translational efficiency of either reporter mRNA, independent of the amount of secondary structure in their respective 5´-UTRs. The increases in translational efficiency produced by Mnk1 occurred in association with corresponding decreases in mRNA levels. These findings indicate that the positive effect of eIF4E phosphorylation on translational efficiency in adult cardiocytes is coupled with the stability of mRNA.

scholarly journals Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

2005 ◽  
Vol 280 (16) ◽  
pp. 15518-15525 ◽  
Author(s):  
Diane E. Handy ◽  
Yufeng Zhang ◽  
Joseph Loscalzo
Keyword(s):  
Glutathione Peroxidase ◽  
Untranslated Region ◽  
Stop Codon ◽  
Vascular Dysfunction ◽  
Lipid Peroxides ◽  
Mrna Levels ◽  
Coding Region ◽  
Sv40 Promoter ◽  
Secis Element

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

scholarly journals Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Ching-Fang Lai ◽  
Chih-Ying Chen ◽  
Lo-Chun Au
Keyword(s):  
Gene Silencing ◽  
Thermodynamic Stability ◽  
Untranslated Region ◽  
Stop Codon ◽  
Secondary Structures ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Post Transcriptional Gene Silencing ◽  
Flanking Sequences

Small interfering RNAs (siRNAs) are applied for post-transcriptional gene silencing by binding target mRNA. A target coding region is usually chosen, although the3′-untranslated region (3′-UTR) can also be a target. This study elucidates whether the coding region or3′-UTR elicits higher repression. pFLuc and pRLuc are two reporter plasmids. A segment ofFLucgene was PCR-amplified and inserted behind the stop codon of theRLucgene of the pRLuc. Similarly, a segment ofRLucgene was inserted behind the stop codon ofFLuc. Two siFLuc and two siRLuc were siRNAs designed to target the central portions of these segments. Therefore, the siRNA encountered the same targets and flanking sequences. Results showed that the two siFLuc elicited higher repression when theFLucsegment resided in the coding region. Conversely, the two siRLuc showed higher repression when theRLucsegment was in the3′-UTR. These results indicate that both the coding region and the3′-UTR can be more effective targets. The thermodynamic stability of the secondary structures was analyzed. The siRNA elicited higher repression in the coding region when the target configuration was stable, and needed to be solved by translation. A siRNA may otherwise favor the target at3′-UTR.

scholarly journals In Yeast, the 3′ Untranslated Region or the Presequence ofATM1 Is Required for the Exclusive Localization of Its mRNA to the Vicinity of Mitochondria

2000 ◽  
Vol 20 (21) ◽  
pp. 7881-7892 ◽  
Author(s):  
M. Corral-Debrinski ◽  
C. Blugeon ◽  
C. Jacq
Keyword(s):  
Untranslated Region ◽  
Mrna Localization ◽  
Rna Localization ◽  
Yeast Cells ◽  
Mitochondrial Proteins ◽  
Coding Region ◽  
Gene Encoding ◽  
Mitochondrial Inner Membrane ◽  
Biochemical Analyses ◽  
Reporter Mrna

ABSTRACT We isolated mitochondria from Saccharomyces cerevisiaeto selectively study polysomes bound to the mitochondrial surface. The distribution of several mRNAs coding for mitochondrial proteins was examined in free and mitochondrion-bound polysomes. Some mRNAs exclusively localize to mitochondrion-bound polysomes, such as the ones coding for Atm1p, Cox10p, Tim44p, Atp2p, and Cot1p. In contrast, mRNAs encoding Cox6p, Cox5a, Aac1p, and Mir1p are found enriched in free cytoplasmic polysome fractions. Aac1p and Mir1p are transporters that lack cleavable presequences. Sequences required for mRNA asymmetric subcellular distribution were determined by analyzing the localization of reporter mRNAs containing the presequence coding region and/or the 3′-untranslated region (3′UTR) of ATM1, a gene encoding an ABC transporter of the mitochondrial inner membrane. Biochemical analyses of mitochondrion-bound polysomes and direct visualization of RNA localization in living yeast cells allowed us to demonstrate that either the presequence coding region or the 3′UTR ofATM1 is sufficient to allow the reporter mRNA to localize to the vicinity of the mitochondrion, independently of its translation. These data demonstrate that mRNA localization is one of the mechanisms used, in yeast, for segregating mitochondrial proteins.

scholarly journals Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3' untranslated region.

1989 ◽  
Vol 108 (2) ◽  
pp. 729-736 ◽  
Author(s):  
S W Hennessy ◽  
B A Frazier ◽  
D D Kim ◽  
T L Deckwerth ◽  
D M Baumgartel ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Cultured Cells ◽  
Coding Region ◽  
Insertion Element ◽  
Initiator Codon ◽  
Cell Growth And Differentiation ◽  
The Stability ◽  
Interferon Gene ◽  
Degree Of Similarity ◽  
Regulatory Sites

The nucleotide sequence of human thrombospondin (TS) mRNA has been determined from human fibroblast and endothelial cDNAs. The sequence of 5802 bp begins 110 bp upstream from the initiator codon and includes the entire 3' untranslated region (UTR) of the mRNA. The coding region (3510 bp) specifies a protein of 1170 amino acids with all of the known features of the TS subunit (Frazier, W. A. 1987. J. Cell Biol. 105:625-632). The long 3' UTR of 2166 nucleotides is extremely A/T-rich, particularly in the latter half. It contains 37 TATT or ATTT(A) sequences that have been suggested as mediators of the stability of mRNAs for cytokines, lymphokines, and oncogenes (Shaw, G., and R. Kamen. 1986. Cell. 46:659-667). Another unusual feature of the 3' UTR of TS mRNA is a stretch of 42 nucleotides of which 40 are thymidines (uridine in the mRNA) including an uninterrupted sequence of 26 thymidines. This region is flanked by two sets of direct repeats suggesting that it may be an insertion element of retrotranscriptional origin. Comparison of the 3' untranslated region of TS mRNA with the GenBank data base indicates the greatest degree of similarity with an alpha-interferon gene which contains a number of the TATT/ATTT consensus sites. The degree of similarity between the TS and interferon sequences is the same in regions of the interferon gene corresponding to its coding and noncoding regions suggesting that most of the TS 3' UTR may be derived from an interferon gene or pseudogene. The features of the TS mRNA 3' UTR provide a potential explanation for the rapid regulation of TS message observed in cultured cells in response to PDGF and suggest that TS is a member of a group of proteins which are intimately involved in the control of cell growth and differentiation.

scholarly journals An eleven nucleotide section of the 3′-untranslated region is required for perinuclear localization of rat metallothionein-1 mRNA

2005 ◽  
Vol 387 (2) ◽  
pp. 419-428 ◽  
Author(s):  
David NURY ◽  
Hervé CHABANON ◽  
Marilyne LEVADOUX-MARTIN ◽  
John HESKETH
Keyword(s):  
Metal Binding ◽  
Untranslated Region ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mrna Localization ◽  
Coding Region ◽  
Ovary Cells ◽  
Structural Context ◽  
Deletion Mutagenesis ◽  
Localization Signal

Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3′-UTR (3′-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3′-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45–76 of the 3′-UTR, in particular nt 66–76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a β-globin coding region is linked to sequences from the MT1 3′-UTR. This section of the 3′-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.

scholarly journals Identification of a calicivirus isolate of unknown origin

2003 ◽  
Vol 84 (10) ◽  
pp. 2837-2845 ◽  
Author(s):  
Angelika Oehmig ◽  
Mathias Büttner ◽  
Frank Weiland ◽  
William Werz ◽  
Klaus Bergemann ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Coding Region ◽  
Electron Microscopic ◽  
Significant Similarity ◽  
Western Blots ◽  
Reading Frame ◽  
A Minor

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

scholarly journals Translation Inhibition of the Salmonella fliC Gene by the fliC 5′ Untranslated Region, fliC Coding Sequences, and FlgM

2006 ◽  
Vol 188 (12) ◽  
pp. 4497-4507 ◽  
Author(s):  
Valentina Rosu ◽  
Fabienne F. V. Chevance ◽  
Joyce E. Karlinsey ◽  
Takanori Hirano ◽  
Kelly T. Hughes
Keyword(s):  
Rna Structure ◽  
Untranslated Region ◽  
Stop Codon ◽  
Mrna Translation ◽  
Base Change ◽  
Targeted Mutagenesis ◽  
Coding Region ◽  
Stem Loop ◽  
Single Base ◽  
Flic Gene

ABSTRACT The 5′-untranslated region (5′UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5′ end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (−38C:U) in the fliC 5′UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the −38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5′UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the −38C:T motility defect, while flgM mutants defective in anti-σ28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5′UTR (−15G:A) results in a translation defect by itself but, in combination with the −38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.

scholarly journals A Review of Methods to Monitor the Modulation of mRNA Stability

2010 ◽  
Vol 15 (6) ◽  
pp. 609-622 ◽  
Author(s):  
Dominique Cheneval ◽  
Tania Kastelic ◽  
Peter Fuerst ◽  
Christian N. Parker
Keyword(s):  
Mrna Stability ◽  
Posttranscriptional Regulation ◽  
Regulation Of Gene Expression ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Cis Acting ◽  
Novel Approach ◽  
Specific Mrnas ◽  
Steady State Levels ◽  
The Stability

Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human diseases, including cancer, inflammatory disease, and Alzheimer’s. These observations suggest that modulating the stability of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and time-consuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be poorly “drugable.” This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and issues related to this approach.

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