scholarly journals The 5′ Untranslated Region and Gag product of Idefix, a Long Terminal Repeat-Retrotransposon from Drosophila melanogaster, Act Together To Initiate a Switch between Translated and Untranslated States of the Genomic mRNA

2003 ◽  
Vol 23 (22) ◽  
pp. 8246-8254 ◽  
Author(s):  
Carine Meignin ◽  
Jean-Luc Bailly ◽  
Frédérick Arnaud ◽  
Bernard Dastugue ◽  
Chantal Vaury
Keyword(s):  
Drosophila Melanogaster ◽  
Long Terminal Repeat ◽  
Untranslated Region ◽  
Terminal Repeat ◽  
Entry Site ◽  
Independent Manner ◽  
Gag Protein ◽  
Genomic Arrangement

ABSTRACT Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5′UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

Binding of a cellular protein to the gibbon ape leukemia virus enhancer

1987 ◽  
Vol 7 (8) ◽  
pp. 2735-2744
Author(s):  
J P Quinn ◽  
N Holbrook ◽  
D Levens
Keyword(s):  
Long Terminal Repeat ◽  
Specific Binding ◽  
Leukemia Virus ◽  
Cellular Protein ◽  
Terminal Repeat ◽  
Enhancer Activity ◽  
Repeated Element ◽  
Gibbon Ape Leukemia Virus

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

scholarly journals miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D

2017 ◽  
Vol 312 (1) ◽  
pp. C71-C82 ◽  
Author(s):  
Yao Li ◽  
Shengjie Li ◽  
Ping Jin ◽  
Liming Chen ◽  
Fei Ma
Keyword(s):  
Drosophila Melanogaster ◽  
Untranslated Region ◽  
Deletion Mutant ◽  
Ectopic Expression ◽  
Whole Body ◽  
Physiological Processes ◽  
Insulin Producing Cells ◽  
Pupal Size

MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3′-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis.

scholarly journals Evolution of the Equine Infectious Anemia Virus Long Terminal Repeat during the Alteration of Cell Tropism

2005 ◽  
Vol 79 (9) ◽  
pp. 5653-5664 ◽  
Author(s):  
Wendy Maury ◽  
Robert J. Thompson ◽  
Quentin Jones ◽  
Sarahann Bradley ◽  
Tara Denke ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Equine Infectious Anemia Virus ◽  
Terminal Repeat ◽  
Binding Motif ◽  
Cell Tropism ◽  
Transcription Factor Binding Motif ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT Equine infectious anemia virus (EIAV) is a lentivirus with in vivo cell tropism primarily for tissue macrophages; however, in vitro the virus can be adapted to fibroblasts and other cell types. Tropism adaptation is associated with both envelope and long terminal repeat (LTR) changes, and findings strongly suggest that these regions of the genome influence cell tropism and virulence. Furthermore, high levels of genetic variation have been well documented in both of these genomic regions. However, specific EIAV nucleotide or amino acid changes that are responsible for cell tropism changes have not been identified. A study was undertaken with the highly virulent, macrophage-tropic strain of virus EIAVwyo to identify LTR changes associated with alterations in cell tropism. We found the stepwise generation of a new transcription factor binding motif within the enhancer that was associated with adaptation of EIAV to endothelial cells and fibroblasts. An LTR that contained the new motif had enhanced transcriptional activity in fibroblasts, whereas the new site did not alter LTR activity in a macrophage cell line. This finding supports a previous prediction that selection for new LTR genetic variants may be a consequence of cell-specific selective pressures. Additional investigations of the EIAVwyo LTR were performed in vivo to determine if LTR evolution could be detected over the course of a 3-year infection. Consistent with previous in vivo findings, we observed no changes in the enhancer region of the LTR over that time period, indicating that the EIAVwyo LTR was evolutionarily stable in vivo.

scholarly journals Construction and In Vitro Characterization of Attenuated Feline Immunodeficiency Virus Long Terminal Repeat Mutant Viruses

2001 ◽  
Vol 75 (2) ◽  
pp. 1054-1060 ◽  
Author(s):  
Luisa Bigornia ◽  
Kristen M. Lockridge ◽  
Ellen E. Sparger
Keyword(s):  
Long Terminal Repeat ◽  
Virus Replication ◽  
Feline Immunodeficiency Virus ◽  
Terminal Repeat ◽  
Immunodeficiency Virus ◽  
Activation Pathways ◽  
Virus Expression ◽  
Transcriptional Elements

ABSTRACT AP-1- and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiplecis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines.

scholarly journals Essential Domains for Ribonucleoprotein Complex Formation Required for Retrotransposition of Telomere-Specific Non-Long Terminal Repeat Retrotransposon SART1

2006 ◽  
Vol 26 (13) ◽  
pp. 5168-5179 ◽  
Author(s):  
Takumi Matsumoto ◽  
Mitsuhiro Hamada ◽  
Mizuko Osanai ◽  
Haruhiko Fujiwara
Keyword(s):  
Long Terminal Repeat ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Open Reading Frames ◽  
Terminal Repeat ◽  
Ltr Retrotransposons ◽  
C Terminus ◽  
Functional Features

ABSTRACT Non-long terminal repeat (LTR) retrotransposons are major components of the higher eukaryotic genome. Most of them have two open reading frames (ORFs): ORF2 encodes mainly the endonuclease and reverse transcriptase domains, but the functional features of ORF1 remain largely unknown. We used telomere-specific non-LTR retrotransposon SART1 in Bombyx mori and clarified essential roles of the ORF1 protein (ORF1p) in ribonucleoprotein (RNP) formation by novel approaches: in vitro reconstitution and in vivo/in vitro retrotransposition assays using the baculovirus expression system. Detailed mutation analyses showed that each of the three CCHC motifs at the ORF1 C terminus are essential for SART1 retrotransposition and are involved in packaging the SART1 mRNA specifically into RNP. We also demonstrated that amino acid residues 555 to 567 and 285 to 567 in the SART1 ORF1p are crucial for the ORF1p-ORF1p and ORF1p-ORF2p interactions, respectively. The loss of these domains abolishes protein-protein interaction, leading to SART1 retrotransposition deficiency. These data suggest that systematic formation of RNP composed of ORF1p, ORF2p, and mRNA is mainly mediated by ORF1p domains and is a common, essential step for many non-LTR retrotransposons encoding the two ORFs.

scholarly journals Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

2001 ◽  
Vol 21 (8) ◽  
pp. 2826-2837 ◽  
Author(s):  
Arun Venkatesan ◽  
Asim Dasgupta
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Independent Manner ◽  
Internal Ribosome Entry

ABSTRACT We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

scholarly journals Essential Motifs in the 3′ Untranslated Region Required for Retrotransposition and the Precise Start of Reverse Transcription in Non-Long-Terminal-Repeat Retrotransposon SART1

2004 ◽  
Vol 24 (18) ◽  
pp. 7902-7913 ◽  
Author(s):  
Mizuko Osanai ◽  
Hidekazu Takahashi ◽  
Kenji K. Kojima ◽  
Mitsuhiro Hamada ◽  
Haruhiko Fujiwara
Keyword(s):  
Long Terminal Repeat ◽  
Reverse Transcription ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Telomeric Repeat ◽  
Terminal Repeat ◽  
Coding Region ◽  
Stem Loop ◽  
Reading Frame

ABSTRACT Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3′ end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3′ untranslated region (3′ UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3′ UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3′ UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3′ UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3′ region (nt 296 to 461) in the 3′ UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3′ UTR anneal to the bottom strand of (TTAGG) n repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3′ UTR and SART1 open reading frame proteins are supplied in trans.

scholarly journals Binding of a cellular protein to the gibbon ape leukemia virus enhancer.

1987 ◽  
Vol 7 (8) ◽  
pp. 2735-2744 ◽  
Author(s):  
J P Quinn ◽  
N Holbrook ◽  
D Levens
Keyword(s):  
Long Terminal Repeat ◽  
Specific Binding ◽  
Leukemia Virus ◽  
Cellular Protein ◽  
Terminal Repeat ◽  
Enhancer Activity ◽  
Repeated Element ◽  
Gibbon Ape Leukemia Virus

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

scholarly journals In vitro and in vivo identification of structural and sequence elements in the 5′ untranslated region of Ectropis obliqua picorna-like virus required for internal initiation

2006 ◽  
Vol 87 (12) ◽  
pp. 3667-3677 ◽  
Author(s):  
Jie Lu ◽  
Jiamin Zhang ◽  
Xiaochun Wang ◽  
Hong Jiang ◽  
Chuanfeng Liu ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Site Directed Mutagenesis ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Sequence Elements ◽  
Internal Initiation

Ectropis obliqua picorna-like virus (EoPV) is a newly described insect virus that is classified as a putative member of the genus Iflavirus. The virus possesses a large, positive-sense RNA genome encoding a single polyprotein that shares physicochemical properties with those of members of the family Picornaviridae. The 5′ untranslated region (5′ UTR) plays an important role in picornavirus translation initiation, as it contains an internal ribosome entry site (IRES) that mediates cap-independent translation. To investigate translation in EoPV, an extensive range of mutations were engineered within the 5′ UTR and the effects of these changes were examined in vitro and in vivo by using a bicistronic construct. Results showed that deletions within the first 63 nt had little impact on IRES activity, whilst core IRES function was contained within stem–loops C and D, as their removal abrogated IRES activity significantly. In contrast to these findings, removal of stem–loop G containing two cryptic AUGs caused a remarkable increase in IRES activity, which was further investigated by site-directed mutagenesis at these two positions. It was also confirmed that initiation of protein synthesis occurs at AUG6 (position 391–394) and not at the AUG immediately downstream of the polypyrimidine tract. Mutation of the polypyrimidine tract (CCTTTC) had a slight effect on EoPV IRES activity. Furthermore, mutations of the RAAA motif led to a decrease in IRES activity of approximately 40 % in vitro, but these results were not supported by in vivo experiments. In conclusion, this study reveals that the EoPV IRES element is unique, although it has features in common with the type II IRESs.

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