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.

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 Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

1999 ◽  
Vol 19 (1) ◽  
pp. 835-845 ◽  
Author(s):  
Zeng-Feng Wang ◽  
Thomas C. Ingledue ◽  
Zbigniew Dominski ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Translational Control ◽  
Mrna Processing ◽  
Loop Structure ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Stem Loop Structure ◽  
Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

scholarly journals Identification of Tau Stem Loop RNA Stabilizers

2007 ◽  
Vol 12 (6) ◽  
pp. 789-799 ◽  
Author(s):  
Christine P. Donahue ◽  
Jake Ni ◽  
Eriks Rozners ◽  
Marcie A. Glicksman ◽  
Michael S. Wolfe
Keyword(s):  
Small Molecules ◽  
Binding Assay ◽  
Loop Structure ◽  
Stem Loop ◽  
Binding Domains ◽  
Stem Loop Structure ◽  
Exon 10 ◽  
Tau Expression

Alternative splicing of tau exon 10 produces tau isoforms with either 3 (3R) or 4 (4R) repeated microtubule-binding domains. Increased ratios of 4R to 3R tau expression, above the physiological 1:1, leads to neurofibrillary tangles and causes neurodegenerative disease. An RNA stem loop structure plays a significant role in determining the ratio, with decreasing stability correlating with an increase in 4R tau mRNA expression. Recent studies have shown that aminoglycosides are able to bind and stabilize the tau stem loop in vitro, suggesting that other druglike small molecules could be identified and that such molecules might lead to decreased exon 10 splicing in vivo. The authors have developed a fluorescent high-throughput fluorescent binding assay and screened a library of ∼110,000 compounds to identify candidate drugs that will bind the tau stem loop in vitro. In addition, they have developed a fluorescent-based RNA probe to assay the stabilizing effects of candiate drugs on the tau stem loop RNA. These assays should be applicable to the general problem of identifying small molecules that interact with mRNA secondary structures. ( Journal of Biomolecular Screening 2007:789-799)

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

scholarly journals Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

2005 ◽  
Vol 393 (1) ◽  
pp. 373-379 ◽  
Author(s):  
Akio Kanai ◽  
Asako Sato ◽  
Jun Imoto ◽  
Masaru Tomita
Keyword(s):  
Thymidylate Synthase ◽  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
In Vitro Translation ◽  
Luciferase Reporter ◽  
Amino Acid Sequence Similarity ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

Using a stem–loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem–loop structure at a high temperature (75 °C) with an apparent dissociation constant of 0.6 μM. A similar stem–loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem–loop structure. In vitro translation analysis using chimaeric constructs containing the stem–loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem–loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine–Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

scholarly journals Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of oriLyt

2007 ◽  
Vol 81 (13) ◽  
pp. 7077-7085 ◽  
Author(s):  
Kelly S. Colletti ◽  
Kate E. Smallenburg ◽  
Yiyang Xu ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Specific Interaction ◽  
Simian Virus 40 ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Core Proteins ◽  
Essential Region

ABSTRACT Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.

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.

A 3' Stem/Loop Structure of the Chlamydomonas Chloroplast atpB Gene Regulates mRNA Accumulation in vivo

1991 ◽  
Vol 3 (3) ◽  
pp. 285 ◽  
Author(s):  
David B. Stern ◽  
Elaine R. Radwanski ◽  
Karen L. Kindle
Keyword(s):  
Loop Structure ◽  
Mrna Accumulation ◽  
Stem Loop ◽  
Stem Loop Structure

scholarly journals Ebola Virus VP30-Mediated Transcription Is Regulated by RNA Secondary Structure Formation

2002 ◽  
Vol 76 (17) ◽  
pp. 8532-8539 ◽  
Author(s):  
Michael Weik ◽  
Jens Modrof ◽  
Hans-Dieter Klenk ◽  
Stephan Becker ◽  
Elke Mühlberger
Keyword(s):  
Secondary Structure ◽  
Transcription Start Site ◽  
Ebola Virus ◽  
Loop Structure ◽  
Start Site ◽  
Transcription Activator ◽  
Transcription Start ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Gene Start

ABSTRACT The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.

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