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 Transcriptional and Translational Control of the Salmonella fliC Gene

2006 ◽  
Vol 188 (12) ◽  
pp. 4487-4496 ◽  
Author(s):  
Phillip Aldridge ◽  
Joshua Gnerer ◽  
Joyce E. Karlinsey ◽  
Kelly T. Hughes
Keyword(s):  
Translational Control ◽  
Gene Promoter ◽  
Mrna Translation ◽  
Start Codon ◽  
Coding Region ◽  
Flagellin Gene ◽  
Stem Loop ◽  
Single Base ◽  
Flic Gene ◽  
Serovar Typhimurium

ABSTRACT The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5′ untranslated region (5′UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5′UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5′ end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the −10 and −35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5′ end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5′UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

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 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 Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Fatima Alghoul ◽  
Schaeffer Laure ◽  
Gilbert Eriani ◽  
Franck Martin
Keyword(s):  
Molecular Mechanisms ◽  
Stop Codon ◽  
Mrna Translation ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Entry Site ◽  
80S Ribosome ◽  
Stem Loop ◽  
Reading Frame ◽  
Translation Inhibition

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The ‘start-stop’ sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

scholarly journals Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

2021 ◽  
Author(s):  
Fatima Alghoul ◽  
Laure Schaeffer ◽  
Gilbert Eriani ◽  
Franck Martin
Keyword(s):  
Molecular Mechanisms ◽  
Stop Codon ◽  
Mrna Translation ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Entry Site ◽  
80S Ribosome ◽  
Stem Loop ◽  
Reading Frame ◽  
Translation Inhibition

AbstractDuring embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The a11 ‘start-stop’ sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

scholarly journals Characterization of the RNA Components of a Putative Molecular Switch in the 3′ Untranslated Region of the Murine Coronavirus Genome

2004 ◽  
Vol 78 (2) ◽  
pp. 669-682 ◽  
Author(s):  
Scott J. Goebel ◽  
Bilan Hsue ◽  
Todd F. Dombrowski ◽  
Paul S. Masters
Keyword(s):  
Untranslated Region ◽  
Genetic Interaction ◽  
Hepatitis Virus ◽  
Stop Codon ◽  
Rna Virus ◽  
Molecular Switch ◽  
Insertion Mutation ◽  
Loss Of Function ◽  
Stem Loop ◽  
Nucleocapsid Gene

ABSTRACT RNA virus genomes contain cis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3′ untranslated region (3′ UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3′ UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.

scholarly journals The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA

2019 ◽  
Vol 48 (2) ◽  
pp. 830-846 ◽  
Author(s):  
Rita Pinto ◽  
Cathrine B Vågbø ◽  
Magnus E Jakobsson ◽  
Yeji Kim ◽  
Marijke P Baltissen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Rna Structure ◽  
Mrna Translation ◽  
28S Rrna ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reversible Modification ◽  
And Shifts ◽  
And Function

Abstract RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem–loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

scholarly journals Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2915-2919 ◽  
Author(s):  
Y Koda ◽  
H Kimura ◽  
E Mekada
Keyword(s):  
Gastric Mucosa ◽  
Pcr Amplification ◽  
Base Change ◽  
Human Gastric Mucosa ◽  
Coding Region ◽  
Single Base ◽  
Cos Cells ◽  
Lewis Antigen ◽  
Base Substitutions ◽  
Nucleotide Mutation

Abstract The expression of Lewis fucosyltransferase (FT) mRNA was examined in gastric mucosa from two Lewis-positive [Le(+)] and two Lewis-negative [Le(-)] individuals. Northern blot analysis demonstrated that levels of mRNA were similar in both Le(+) and Le(-) gastric mucosa. We isolated the protein-coding region of the Lewis FT cDNA from Le(+) and Le(-) gastric mucosa by polymerase chain reaction (PCR) amplification. The sequence of cDNA from the Le(-) gastric mucosa shows two single-base substitutions of G for T at position 59 and of A for G at position 508 from the A of the initiation codon of cDNA. These substitutions may be the cause of changes in two amino acid residues, Arg for Leu at position 20 and Ser for Gly at position 170 from the N-terminal. To determine whether either or both of these base substitutions is responsible for the Le(-) gene, we constructed chimera cDNAs and expressed them in COS cells. Those COS cells transfected with a chimera cDNA containing a mutation of the 508th nucleotide did not express Lewis antigen, whereas those cells transfected with a chimera cDNA containing the 59th nucleotide mutation expressed Lewis antigen, indicating that a single-base change from G to A at position 508 is responsible for the Le(-) gene. The G to A transition at position 508 created a new site for PvuII endonuclease. The digestion by PvuII endonuclease of PCR products between the 386th and 612th nucleotides of Lewis FT cDNA from one of the Le(-) individuals proved to be homozygous for the PvuII site. However, the other Le(-) individual was heterozygous for the PvuII site, suggesting the presence of other Le(-) allele(s). Thus, we isolated one of the silent Lewis genes (le).

scholarly journals Similar Interactions of the Poliovirus and Rhinovirus 3D Polymerases with the 3′ Untranslated Region of Rhinovirus 14

1999 ◽  
Vol 73 (12) ◽  
pp. 9952-9958 ◽  
Author(s):  
Janet M. Meredith ◽  
Jonathan B. Rohll ◽  
Jeffrey W. Almond ◽  
David J. Evans
Keyword(s):  
Untranslated Region ◽  
Specific Interaction ◽  
Low Frequency ◽  
Coding Region ◽  
Reporter System ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Active Site Cleft ◽  
Plaque Phenotype ◽  
Type 3

ABSTRACT We showed previously that a human rhinovirus 14 (HRV14) 3′ untranslated region (3′ UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835–7844, 1995). This enabled the HRV14 single 3′ UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3′ UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3′ UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3′ UTR of rhinovirus with both poliovirus and rhinovirus polymerases.

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