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1.
Conditional gene expression by controlling translation with tetracycline-binding aptamers 总被引:9,自引:2,他引:7
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We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA–metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5′-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability. 相似文献
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MicroRNAs (miRs) commonly regulate translation from target mRNA 3' untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5' untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3'UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5'UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5'UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5'UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3'UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3'UTR. 相似文献
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Drosophila Sex-lethal (dSXL)-mediated translational repression of male-specific lethal 2 (msl-2) mRNA is essential for X-chromosome dosage compensation. Binding of dSXL to specific sites in both untranslated regions of msl-2 mRNA is necessary for inhibition of translation initiation. We describe the organization of dSXL as a translational regulator and show that the RNA binding and translational repressor functions are contained within the two RRM domains and a C-terminal heptapeptide extension. The repressor function is dormant unless dSXL binds to msl-2 mRNA with its own RRMs, because dSXL tethering via a heterologous RNA-binding peptide does not elicit translational inhibition. We reveal proteins that crosslink to the msl-2 3' untranslated region (3'UTR) and co-immunoprecipitate with dSXL in a fashion that requires its intact repressor domain and correlates with translational regulation. Translation competition and UV-crosslink experiments show that the 3'UTR msl-2 sequences adjacent to dSXL-binding sites are necessary to recruit titratable co-repressors. Our data support a model where dSXL binding to the 3'UTR of msl-2 mRNA activates the translational repressor domain, thereby enabling it to recruit co-repressors in a specific fashion. 相似文献
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Cerny RE Qi Y Aydt CM Huang S Listello JJ Fabbri BJ Conner TW Crossland L Huang J 《Plant molecular biology》2003,52(2):357-369
We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants. RNA sequences called translational operators were inserted at a cap-proximal position in the 5-UTR of mRNAs of two reporter genes, gusor aroA:CP4. Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed. The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells. Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent. A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal. Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.these authors contributed equally to this workthese authors contributed equally to this work 相似文献
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Umehara T Fukuda K Nishikawa F Kohara M Hasegawa T Nishikawa S 《Journal of biochemistry》2005,137(3):339-347
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Delta, was conjugated via an oligo(U) tract to the 3'-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition. 相似文献
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Interaction of proteins with the mRNA for ribosomal protein L1 in Xenopus: structural characterization of in vivo complexes and identification of proteins that bind in vitro to its 5'UTR. 总被引:4,自引:1,他引:3
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Xenopus r-protein mRNAs are known to be coordinately regulated at the translational level. To find out if RNA/protein interactions are involved in this control mechanism, we have characterized the particles containing the translationally repressed rp-mRNA and we have investigated the proteins that specifically bind to this type of mRNA. By sedimentation analysis and isopycnic centrifugation we have found that the repressed rp-mRNAs are assembled in slow sedimenting complexes where the RNA is prevalent over the protein mass (2.3 to 1). This composition is maintained also after in vitro reconstitution of the particle. We carried out also a detailed analysis of in vitro RNA/protein complex formation by focusing our attention on the 5'UTR, very similar in different rp-mRNAs and important in the translational regulation. We describe specific interactions of L1 mRNA with four proteins. The binding site of two of them, 57 kD and 47 kD, is in the typical pyrimidine sequence at the 5' end and is position dependent. Proteins of the same size interact also with the analogous region of r-protein S1 and L14 mRNA, not with unrelated RNAs. Binding of two other proteins, 31 kD and 24 kD, in the downstream region of the 5'UTR was also observed. The most evident 57 kD protein has been partially purified. Although the binding of these proteins to the r-protein mRNA 5'UTR is specific, their involvement in the translation regulation remains to be proved. 相似文献
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Nishikawa F Kakiuchi N Funaji K Fukuda K Sekiya S Nishikawa S 《Nucleic acids research》2003,31(7):1935-1943
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High affinity RNA for mammalian initiation factor 4E interferes with mRNA-cap binding and inhibits translation 总被引:2,自引:0,他引:2
The eukaryotic translation initiation factor 4F (eIF4F) consists of three polypeptides (eIF4A, eIF4G, and eIF4E) and is responsible for recruiting ribosomes to mRNA. eIF4E recognizes the mRNA 5'-cap structure (m7GpppN) and plays a pivotal role in control of translation initiation, which is the rate-limiting step in translation. Overexpression of eIF4E has a dramatic effect on cell growth and leads to oncogenic transformation. Therefore, an inhibitory agent to eIF4E, if any, might serve as a novel therapeutic against malignancies that are caused by aberrant translational control. Along these lines, we developed two RNA aptamers, aptamer 1 and aptamer 2, with high affinity for mammalian eIF4E by in vitro RNA selection-amplification. Aptamer 1 inhibits the cap binding to eIF4E more efficiently than the cap analog m7GpppN or aptamer 2. Consistently, aptamer 1 inhibits specifically cap-dependent in vitro translation while it does not inhibit cap-independent HCV IRES-directed translation initiation. The interaction between eIF4E and eIF4E-binding protein 1 (4E-BP1), however, was not inhibited by aptamer 1. Aptamer 1 is composed of 86 nucleotides, and the high affinity to eIF4E is affected by deletions at both termini. Moreover, relatively large areas in the aptamer 1 fold are protected by eIF4E as determined by ribonuclease footprinting. These findings indicate that aptamers can achieve high affinity to a specific target protein via global conformational recognition. The genetic mutation and affinity study of variant eIF4E proteins suggests that aptamer 1 binds to eIF4E adjacent to the entrance of the cap-binding slot and blocks the cap-binding pocket, thereby inhibiting translation initiation. 相似文献
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A tetracycline-binding RNA aptamer 总被引:5,自引:0,他引:5
Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit. 相似文献
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Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline. 相似文献
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Identification of cis-acting sequences required for translational autoregulation of the ermC methylase. 总被引:7,自引:5,他引:2
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ermC methylase gene expression has been shown to be limited by translational autorepression, presumably due to methylase binding to ermC mRNA. It was found that this repression occurs in trans, yielding a 50% reduction in translation of an ermC-lacZ fusion mRNA. We investigated the ermC mRNA sequences required for translational repression in vivo. A series of deletions identified sequences in the 5' regulatory region that were required for translational repression. These included sequences of the 5' stem-loop structure that were not required for induction, as well as some that were required. The implications of these results for regulation are discussed. 相似文献
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We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells. 相似文献
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The importance of the 3'-untranslated region in the translational control of ferritin mRNA 总被引:11,自引:0,他引:11
L F Dickey Y H Wang G E Shull I A Wortman E C Theil 《The Journal of biological chemistry》1988,263(7):3071-3074