scholarly journals Influence of the 5′-proximal elements of the 5′-untranslated region of classical swine fever virus on translation and replication

2011 ◽  
Vol 92 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Ming Xiao ◽  
Yujing Wang ◽  
Zailing Zhu ◽  
Chengli Ding ◽  
Jialin Yu ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Untranslated Region ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Loop Structure ◽  
Terminal Sequence ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Stem Loop Structure

The 5′-terminal sequence spanning nt 1–29 of the 5′-untranslated region of classical swine fever virus (CSFV) forms a 5′-proximal stem–loop structure known as domain Ia. Deletions and replacement mutations were performed to examine the role of this domain. Deletion of the 5′-proximal nucleotides and disruption of the stem–loop structure greatly increased internal ribosome entry site-mediated translation but abolished the replication of the replicons. Internal deletions resulting in a change in the size of the loop of domain Ia, and even removal of the entire domain, did not substantially change the translation activity, but reduced the replication of CSFV replicons provided the replicons contained the extreme 5′-GUAU terminal sequence. Internal replacements leading to a change in the nucleotide sequence of the loop did not alter the translation and replication activities of the CSFV RNA replicon, and did not influence the rescue of viruses and growth characteristics of new viruses. These results may be important for our understanding of the regulation of translation, replication and encapsidation in CSFV and other positive-sense RNA viruses.

scholarly journals Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

2009 ◽  
Vol 90 (12) ◽  
pp. 2923-2928 ◽  
Author(s):  
Ming Xiao ◽  
Yujing Wang ◽  
Zailing Zhu ◽  
Jialin Yu ◽  
Lingzhu Wan ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Internal Ribosome Entry Site ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Border Disease Virus ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Diarrhea Virus ◽  
Border Disease

An internal ribosome entry site (IRES) present in the 5′ untranslated region (UTR) promotes translation of classical swine fever virus (CSFV) genomes. Using an in vitro system with monocistronic reporter RNA containing the CSFV 5′UTR, this study found that CSFV NS5A decreased CSFV IRES-mediated translation in a dose-dependent manner. Deletion analysis showed that the region responsible for repressing CSFV IRES activity might cover aa  390–414, located in the C-terminal half of CSFV NS5A. Triple and single alanine-scanning mutagenesis revealed that the inhibitory effect on CSFV IRES-directed translation mapped to the K399, T401, E406 and L413 residues of NS5A. These important amino acids were also found to be present in the NS5A proteins of bovine viral diarrhea virus (BVDV)-1, BVDV-2, border disease virus and hepatitis C virus, indicating that NS5A may play an important role in the switch from translation to replication in these viruses.

scholarly journals Role of a Stem-Loop Structure inHelicobacter pylori cagATranscript Stability

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
John T. Loh ◽  
Aung Soe Lin ◽  
Amber C. Beckett ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Steady State ◽  
Untranslated Region ◽  
Loop Structure ◽  
Stem Loop ◽  
Content Type ◽  
Transcript Stability ◽  
Protein Levels ◽  
Caga Protein ◽  
Stem Loop Structure ◽  
Steady State Levels

ABSTRACTHelicobacter pyloriCagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation amongH. pyloristrains in the steady-state levels of CagA and that a strain-specific motif downstream of thecagAtranscriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. ThecagA5′ untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop oncagAtranscript levels andcagAmRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both thecagAtranscript and the CagA protein. Additionally, these mutations resulted in a decreasedcagAmRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-statecagAtranscript and CagA protein levels but did not affectcagAtranscript stability.cagAtranscript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augmentcagAtranscript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in thecagA5′ untranslated region influence the levels ofcagAexpression.

scholarly journals Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication

2007 ◽  
Vol 88 (6) ◽  
pp. 1738-1747 ◽  
Author(s):  
Patrícia A. G. C. Silva ◽  
Richard Molenkamp ◽  
Tim J. Dalebout ◽  
Nathalie Charlier ◽  
Johan H. Neyts ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Molecular Mechanisms ◽  
Yellow Fever Virus ◽  
Animal Cell ◽  
Fever Virus ◽  
Loop Structure ◽  
Animal Cells ◽  
Pn Sequence ◽  
Stem Loop ◽  
Stem Loop Structure

The pentanucleotide (PN) sequence 5′-CACAG-3′ at the top of the 3′ stem–loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.

scholarly journals Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142 ◽  
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

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.

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

A small predicted stem-loop structure mediates oocyte localization of Drosophila K10 mRNA

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3809-3818 ◽  
Author(s):  
T.L. Serano ◽  
R.S. Cohen
Keyword(s):  
Late Stage ◽  
Untranslated Region ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Loop Structure ◽  
Base Pairing ◽  
Mrna Transport ◽  
Nucleotide Substitutions ◽  
Stem Loop ◽  
Stem Loop Structure

The establishment of dorsoventral polarity in the Drosophila oocyte and future embryo is dependent on the efficient transport of K10 mRNA from nurse cells into the oocyte. To investigate the cis-requirements of K10 mRNA transport, we used a transgenic fly assay to analyze the expression patterns of a series of K10 deletion variants. Such studies identify a 44 nucleotide sequence within the K10 3′ untranslated region that is required and sufficient for K10 mRNA transport and subsequent localization to the oocyte's anterior cortex. An inspection of the 44 nucleotide transport/localization sequence (TLS) reveals a strong potential for the formation of a stem-loop secondary structure. Nucleotide substitutions that interfere with the predicted base-pairing of the TLS block mRNA transport and anterior localization. Conversely, mutations that alter the base composition of the TLS while maintaining predicted base-pairing do not block mRNA transport or anterior localization. We conclude that K10 mRNA transport and anterior localization is mediated by a 44 nucleotide stem-loop structure. A similar putative stem-loop structure is found in the 3′ untranslated region of the Drosophila orb mRNA, suggesting that the same factors mediate the transport and anterior localization of both K10 and orb mRNAs. Apart from orb, the K10 TLS is not found in any other localized mRNA, raising the possibility that the transport and localization of other mRNAs, e.g., bicoid, oskar and gurken, are mediated by novel sets of cis- and trans-acting factors. Moreover, we find that the K10 TLS overrides the activity of oskar cis-regulatory elements that mediate the late stage movement of the mRNA to the posterior pole. We propose the existence of a family of cis-regulatory elements that mediate mRNA transport into the oocyte, only some of which are compatible with the elements that mediate late stage movements.

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