A Secondary Structure in the 5' Untranslated Region ofadhEmRNA Required for RNase G-Dependent Regulation

2013 ◽  
Vol 77 (12) ◽  
pp. 2473-2479 ◽  
Author(s):  
Kazutaka ITO ◽  
Kohshin HAMASAKI ◽  
Aya KAYAMORI ◽  
Phuong Anh Thi NGUYEN ◽  
Kaoru AMAGAI ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Rnase G

scholarly journals Role of the 5′-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine

1994 ◽  
Vol 302 (3) ◽  
pp. 765-772 ◽  
Author(s):  
L M Shantz ◽  
R Viswanath ◽  
A E Pegg
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Fold Increase ◽  
Negative Regulator ◽  
Mrna Levels ◽  
Coding Region ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Synthase Inhibitor

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5′-untranslated region (5′UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5′UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5′UTR, as well as chimaeric constructs in which the AdoMetDC 5′UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5′UTR, the AdoMetDC protein-coding region and the 3′UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5′UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5′UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5′ end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5′UTR that may include but is not entirely due to the internal ORF.

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 HuR Uses AUF1 as a Cofactor To Promote p16INK4 mRNA Decay

2010 ◽  
Vol 30 (15) ◽  
pp. 3875-3886 ◽  
Author(s):  
Na Chang ◽  
Jie Yi ◽  
Gaier Guo ◽  
Xinwen Liu ◽  
Yongfeng Shang ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Mrna Decay ◽  
Loop Structure ◽  
Wild Type ◽  
Stem Loop ◽  
Human Diploid Fibroblasts ◽  
Stem Loop Structure ◽  
Chimeric Transcripts ◽  
P16 Expression

ABSTRACT In this study, we show that HuR destabilizes p16INK4 mRNA. Although the knockdown of HuR or AUF1 increased p16 expression, concomitant AUF1 and HuR knockdown had a much weaker effect. The knockdown of Ago2, a component of the RNA-induced silencing complex (RISC), stabilized p16 mRNA. The knockdown of HuR diminished the association of the p16 3′ untranslated region (3′UTR) with AUF1 and vice versa. While the knockdown of HuR or AUF1 reduced the association of Ago2 with the p16 3′UTR, Ago2 knockdown had no influence on HuR or AUF1 binding to the p16 3′UTR. The use of EGFP-p16 chimeric reporter transcripts revealed that p16 mRNA decay depended on a stem-loop structure present in the p16 3′UTR, as HuR and AUF1 destabilized EGFP-derived chimeric transcripts bearing wild-type sequences but not transcripts with mutations in the stem-loop structure. In senescent and HuR-silenced IDH4 human diploid fibroblasts, the EGFP-p16 3′UTR transcript was more stable. Our results suggest that HuR destabilizes p16 mRNA by recruiting the RISC, an effect that depends on the secondary structure of the p16 3′UTR and requires AUF1 as a cofactor.

scholarly journals Secondary structure of dengue virus type 4 3′ untranslated region: impact of deletion and substitution mutations

2006 ◽  
Vol 87 (11) ◽  
pp. 3291-3296 ◽  
Author(s):  
Tammy A. Romero ◽  
Ebenezer Tumban ◽  
Jeongwon Jun ◽  
William B. Lott ◽  
Kathryn A. Hanley
Keyword(s):  
Secondary Structure ◽  
Dengue Virus ◽  
Untranslated Region ◽  
Modes Of Transmission ◽  
The Core ◽  
Homologous Sequences ◽  
Langat Virus ◽  
Computer Based ◽  
Substitution Mutations

Several studies have generated computer-based predictions of secondary structure of the 3′ untranslated region (UTR) of Dengue virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 3′ UTR with nuclease maps of this region. Maps and predictions were largely consistent. Maps supported the existence of previously predicted pseudoknots and identified putative regions of dynamic folding. Additionally, this study investigated previously identified conserved elements in the flavivirus 3′ UTR that differ among viruses with different modes of transmission. Specific regions of mosquito-borne DEN type 4 were either deleted or replaced with homologous sequences from tick-borne Langat virus. All of these mutations caused substantial distortion of secondary structure, yet viruses carrying these mutations were viable.

scholarly journals The Leader RNA of Coronavirus Mouse Hepatitis Virus Contains an Enhancer-Like Element for Subgenomic mRNA Transcription

2000 ◽  
Vol 74 (22) ◽  
pp. 10571-10580 ◽  
Author(s):  
Yicheng Wang ◽  
Xuming Zhang
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Consensus Sequence ◽  
Mouse Hepatitis Virus ◽  
Mrna Transcription ◽  
Rt Pcr ◽  
Reporter System ◽  
Rna Transcription ◽  
Subgenomic Rna

ABSTRACT While the 5′ cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize thecis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of thecis-acting signal for replication in the 5′ untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5′-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5′ untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5′ cis-acting sequence regulates subgenomic RNA transcription. The 5′-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines thecis-acting transcription signal at the 5′ end of the DI genome.

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