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The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure
Introduction
Materials And Methods
Plasmid constructs
In vitro transcription reactions
Preparation of nuclear extracts and purified CPSF/CstF
EMSA
Results
Stable RNA structure interferes with the binding of polyadenylation factors
Revertants of the stabilized mutant hairpin rescue binding of polyadenylation factors
The USE enhancer specifically overcomes the structure-mediated repression of the HIV-1 poly(A) site
Leader-specific inhibition of the HIV-1 poly(A) site
Discussion
Acknowledgements
References
The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure
ABSTRACT
INTRODUCTION
Polyadenylation is one of the mRNA maturation steps in eukaryotic cells. Poly(A) tails can endow critical properties on the mRNA, such as stability, nuclear export and translatability (1-5). Polyadenylation involves cleavage of the primary transcript and subsequent addition of ~250 adenosine ribonucleotides (6-11). Polyadenylation of mRNA precursors depends on specific RNA sequences that are recognized by nuclear protein factors (6,12-14). At least two sequence elements define a poly(A) site. The AAUAAA hexamer is the almost invariant poly(A) signal that is positioned ~15 nt upstream of the site of cleavage. A poorly conserved GU-rich or U-rich element is usually positioned 20-30 nt downstream of the hexamer (15-21). These two elements form the core poly(A) site. Additional upstream enhancer (USE) elements have been described, in particular for viral RNAs (22-29). Although generally U-rich, these enhancer elements exhibit little sequence or structural similarity. The efficiency of RNA processing has been shown to correlate with the binding of polyadenylation factors to these signals (30).
The two key components responsible for the specific recognition of poly(A) sites are the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulation factor (CstF) (6). CPSF consists of four polypeptide subunits with molecular weights of 160, 100, 73 and 30 kDa (31,32). CstF consists of three polypeptides of 77, 64 and 50 kDa. CPSF binds specifically to the AAUAAA signal and CstF binds to the downstream enhancer element (13,32-35). The presence of CPSF is required for efficient binding of CstF, and the interaction of CPSF with the RNA is stabilized by CstF. The stability of this RNA-CPSF-CstF ternary complex has been shown to correlate with the efficiency of mRNA processing (30). These factors form a 3[prime]-processing complex upon association of the cleavage factors CFI and CFII and the 82 kDa poly(A) polymerase (PAP). It is generally assumed that CstF and the cleavage factors leave the complex after the cleavage reaction. CPSF and PAP remain bound to the RNA precursor and facilitate the addition of the poly(A) tail.
The HIV-1 retroviral genome contains a 97 nt repeat region (R) that forms both the extreme 5[prime] and 3[prime] ends of the mature RNA. Because the poly(A) signal is encoded within the R region, it is present at both ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5[prime] poly(A) signal or enhance usage of the 3[prime] signal. HIV-1 has been reported to have both regulatory features (Fig.
Figure 1. Schematic of transcripts that mimic the 5[prime] or 3[prime] end of HIV-1 RNA. The HIV-1 DNA provirus is shown at the top, with a close up of the long terminal repeats (LTRs). The LTRs are divided in the U3, R and U5 regions. Nucleotide numbers refer to positions on the genomic RNA, with +1 being the capped G residue. The primary transcript is initiated at the U3/R border within the 5[prime] LTR, and polyadenylation occurs at the R/U5 border within the 3[prime] LTR. The R region encodes the TAR hairpin and the poly(A) hairpin, and the latter structure contains the AAUAAA poly(A) signal (indicated by an arrow-head). The major SD (closed box) in the untranslated leader region has been reported to inhibit polyadenylation at the 5[prime] poly(A) site (-sign). The U3 region encodes the USE (open box) that has been reported to stimulate polyadenylation at the 3[prime] poly(A) site (+ sign). The T7-transcripts used in EMSA in this study are depicted below. Transcripts that mimic the 5[prime] poly(A) site start at position +1 and have variable 3[prime] ends (position +134, +202, +245, +290 or +368). Transcripts that mimic the 3[prime] poly(A) site start at position -54, thereby including the USE enhancer. Only the -54/+134 RNA represents the true 3[prime]-context. All other transcripts with an extended 3[prime] end contain sequences derived from the 5[prime] leader region, and thus represent hybrid 5[prime]/3[prime] transcripts. The 180R transcripts fuse non-HIV sequences (indicated by a wavy line) to the HIV-1 poly(A) signal. Previous studies indicated that the poly(A) signal of the HIV-1 mRNA is located within a stem-loop structure that we termed the poly(A) hairpin (42) (Fig. Figure 2. RNA structure of the wild-type poly(A) hairpin and the mutants/revertants used in this study. Nucleotide numbers refer to positions on the wild-type (wt) genomic RNA, with +1 being the capped G residue. The poly(A) signal AAUAAA is marked by shading. Structure prediction and free-energy minimization were performed with the MFOLD program (70) in the GCG package, and the thermodynamic stability calculated for 37°C is presented below the structures (kcal/mol). We note that there is considerable biochemical, phylogenetic and virological evidence for the existence of this stem-loop structure (42-44). In mutant A, the hairpin is stabilized by deletion (black triangle) of two bulged nucleotides and one nucleotide substitution (boxed). In mutants B and C, destabilizing mutations were introduced into the left- and right-hand side of the stem, respectively. A2, A4 and A7 are revertants of mutant A and the mutations that mediate the reversion phenotype are marked by black boxes. The HIV-1 sequences used in this study are derived from the infectious molecular clone pLAI (54). Several mutations were introduced into the poly(A) hairpin of the pBlue-5[prime]LTR subclone as described previously (44). Revertant viruses were obtained in prolonged virus culture experiments, and part of their genome was subcloned in the pBlue-5[prime]LTR plasmid (55). pBlue-5[prime]LTR contains an XbaI-ClaI fragment of pLAI, which includes the complete 5[prime] LTR and downstream sequences up to 40 nt downstream of the startcodon of the Gag open reading frame (56). pBlue-3[prime]LTR contains the complete 3[prime] LTR (up to LAI position +183) as an XhoI-BglI fragment (57), with downstream non-HIV sequences derived from the pBR322 plasmid. Control transcripts were synthesized from plasmids that contain the poly(A) site of the adenovirus-2 L3 mRNA, with either the wild-type poly(A) signal AAUAAA (Adwt) or the mutant AAGAAA motif (Admut) (58). The pBlue-5[prime]LTR and pBlue-3[prime]LTR plasmids were used as template for PCR amplification and subsequent in vitro transcription. All nucleotide positions relate to the position on the viral RNA genome, with +1 being the transcriptional start site, and nucleotide positions in the 3[prime]R region will be numbered according to the corresponding 5[prime]R position. The sense primers T7-1 and T7-2 contain the T7 RNA polymerase promoter sequence (underlined in Table 1) and will anneal to the HIV-1 sequences starting at positions -54 and +1, respectively. The antisense primers used for amplification of different HIV-1 fragments are TU5, lys21, BB3, ADSD and AD-AUG, and their positions on the HIV-1 RNA genome are indicated in Table 1. The AatII primer was used for amplification of HIV-1 sequences fused to downstream non-HIV sequences on plasmid pBlue-3[prime]LTR. The PCR reactions were performed in PCR buffer (20 mM Tris-HCl pH 8.3, 2 mM MgCl2, 50 mM KCl, 0.1 mg/ml BSA) containing 100 ng of each primer, 10 mM of each dNTP, 1.5 U Taq polymerase and 10 ng plasmid. adenoviral transcripts were synthesized with T3 RNA polymerase on DraI-linearized plasmids (58). The HIV-1 PCR fragments and the linearized adenovirus plasmid constructs were phenol-extracted, precipitated and dissolved in water. The in vitro transcription reaction was performed in 10 µl transcription buffer (40 mM Tris pH 7.5, 2 mM spermidine, 10 mM DTT and 12 mM MgCl2) containing ~0.5 µg DNA template, 20 µCi [[alpha]-32P]UTP (3000 Ci/mmol), 0.06 µmol ATP, 0.06 µmol GTP, 0.06 µmol CTP, 0.02 µmol UTP, 10 U T7 or T3 RNA polymerase (Boehringer Mannheim) and 20 U RNase inhibitor (Boehringer Mannheim). RNA synthesis was continued for 2 h at 37°C, and then the template was removed by addition of 1 µl (2 U) DNase I (Boehringer Mannheim) for 30 min at 37°C. The radiolabeled transcripts were separated on a 6% acryl:bisacrylamide (19:1)/7 M urea gel. The gel fragments containing the full-size transcripts were isolated, and incubated in 450 µl elution buffer (0.3 M NaAc pH 5.2) and 100 µl phenol:chloroform:isoamylalcohol (25:24:1) at 37°C for 15 h. After phenol-extraction, 900 µl ethanol (96%) were added for precipitation of the RNA transcripts. The transcripts were dissolved in renaturation buffer (10 mM Tris pH 7.5, 100 mM NaCl), heated for 2 min at 85°C, and then cooled slowly to room temperature, and stored at -20°C. Table 1. HeLa cells (50 × 106 cells) were grown to 60% confluency in 90 mm dishes in Dulbecco's modified Eagle`s medium containing 8% fetal calf serum (FCS), 100 U of penicillin and 100 µg/ml of streptomycin at 37°C and 5% CO2. The cells were washed once with prewarmed (37°C) PBS and put on ice, and then 2 ml of ice-cold PBS were added. The cells were collected by scraping with a rubber policeman, and washed twice with cold PBS. The cells were pelleted for 10 min in a Sorvall centrifuge at 2000 g. All further steps were performed in the cold room. The pellet was dissolved in 0.5 ml nuclear extract buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40), incubated on ice for 1 min, and centrifuged at 120 g for 5 min to pellet the nuclei. The pellet was resuspended in one packed cell volume of lysis buffer I [20 mM HEPES pH 7.9, 600 mM KCl, 0.2 mM EDTA and 0.2 mM EGTA, 2 mM Pefabloc (Boehringer Mannheim), 2 mM DTT]. The extract was incubated on ice for 30 min and subsequently cleared by centrifugation at 10 000 g for 10 min. The supernatant was collected and one packed cell volume lysis buffer II (20 mM HEPES pH 7.9, 20% glycerol, 0.2 mM EDTA and 0.2 mM EGTA) was added. The nuclear extract was frozen in liquid nitrogen and stored at -70°C. Protein concentrations were determined by the Bradford Method (59). For RNA binding assays, we used 3 µl of the HeLa nuclear extract (4 mg/ml). Purified CPSF and CstF were obtained from HeLa cells as fractions IIIC and IA, respectively, according to the protocol of Åström et al. (60). We used 9 µl CPSF (0.6 mg/ml) and 2 µl CstF (1.1 mg/ml) per RNA binding reaction. Complexes between HIV-1 RNA and polyadenylation factors were assembled as described (27) in 25 µl band-shift buffer [16.5 mM HEPES pH 7.9, 83 mM KCl, 8.3% glycerol, 1% polyvinylalcohol (Sigma), 0.016% NP-40, 16 µg/ml tRNA (Escherichia coli, Boehringer Mannheim), 0.17 mM EDTA, 0.41 mM DTT]. A standard amount of the 32P-labeled transcript was used per assay (1 nCi). The samples were incubated with the purified polyadenylation factors or nuclear extract for 10 min at 30°C, placed on ice and then heparin (Sigma) was added to 5 mg/ml. The samples were analyzed on a non-denaturing 3% acryl:bisacrylamide (19:1)/0.25× TBE with 5% glycerol. Electrophoresis was performed for 3 h at 200 V at room temperature. The gel was dried and signals were visualized and quantitated by PhosphorImager (Molecular Dynamics). It was difficult to compare the activity in the poly(A) complex with that of free RNA because of the formation of an aspecific RNA-protein complex of low molecular weight that is seen as a smear in most EMSA experiments. Therefore, we used equal counts of the different transcripts with the same specific activity per EMSA sample and thus per lane, and we simply measured the counts of the poly(A) complex. The efficiency of poly(A) complex of the wild-type -54/+134 transcript was set at 100% for each experiment. We analyzed the effect of RNA secondary structure of the HIV-1 poly(A) site on the binding of polyadenylation factors. To do this, wild-type and mutant transcripts were synthesized that mimic the 3[prime]-context within the HIV-1 genome (Fig. We first compared the migration of several 32P-labeled transcripts in the absence (Fig. Table 2. Figure 3. Stabilization of the poly(A) hairpin occludes binding of polyadenylation factors. EMSA was performed with adenoviral transcripts and 3[prime]-like HIV-1 RNAs (position -54/+134) in combination with HeLa nuclear extract (lanes 1-6). The mock-incubated RNA samples are included for comparison (lanes 7-12). The transcript that was used is indicated above the lanes. The adenoviral transcripts have either the wild-type AAUAAA poly(A) signal (Adwt) or the mutant AAGAAA signal (Admut). The wild-type and mutant poly(A) hairpin in the HIV-1 RNAs are shown in detail in Figure 2. The position of the free RNA and the RNA-protein complex is indicated on the left. A representative experiment is shown, but similar results were obtained in at least six independent assays. We next tested the wild-type and mutant HIV-1 transcripts in EMSA with the purified CPSF and CstF polyadenylation factors (Fig. Figure 4. The interaction of HIV-1 transcripts with purified polyadenylation factors. The wild-type and mutant HIV transcripts (3[prime]-like, -54/+134) were incubated with either purified CPSF and CstF (lanes 1-4) or HeLa nuclear extract (lanes 5-8). The position of free RNA and the poly(A) complex is indicated on the left. Similar results were obtained in a duplicate experiments with the purified factors, and the experiment with HeLa nuclear extract was repeated at least six times, with the same results. Another difference in EMSA with the two sources of poly-adenylation factors is the gel mobility of the poly(A) complexes, suggesting a difference in the composition of the assembled RNA-protein complexes. The complex made with CPSF and CstF migrated faster than the complex made with total nuclear extract (compare for instance lanes 4 and 5 of Fig. The combined EMSA results with the control adenoviral transcripts and the purified factors indicate that we are able to assemble poly(A) complexes on HIV-1 RNA, and this reaction is exquisitely sensitive to stable RNA structure. RNA structure seems to inhibit the initial step of polyadenylation, that is formation of the RNA-CPSF-CstF complex. The negative effect of RNA structure was dominant in case of the A mutant template with a stabilized hairpin. The wild-type HIV-1 template was suboptimal compared with the mutants that contain a destabilized hairpin in EMSA with purified factors, but full activity was measured in a HeLa cell extract that allows for the formation of larger, presumably more mature poly(A) complexes. Thus, whereas binding of the individual CPSF or CstF factors to the HIV-1 AAUAAA signal is inefficient, the increased affinity of the more mature poly(A) complex for the RNA is able to effectively compete with the repressive RNA structure. The A mutant template with the stabilized poly(A) hairpin was a poor template for assembly of the poly(A) complex in all experimental settings. Because this mutant contains three nucleotide changes compared with the wild-type (Fig. EMSA was performed with the wild-type, mutant and revertant transcripts (Fig. Figure 5. The inability of the stabilized hairpin to bind polyadenylation factors is rescued by mutations that weaken the structure. EMSA with 5[prime]-like HIV-1 transcripts (-54/+134) and HeLa nuclear extract (lanes 1-5). The poly(A) mutant and revertant RNAs that were used are indicated on top of the lanes. Mock RNA samples are present in lanes 6-10. The position of the free RNA and the poly(A) complex is indicated on the left. So far we have tested HIV-1 transcripts that start at position -54 in the U3 region and end at position +134 (Fig. Figure 6. EMSA with HIV-1 transcripts that mimic either the 5[prime] or 3[prime] poly(A) site. EMSA was performed with HeLa nuclear extract and HIV-1 transcripts that either mimic the 3[prime]-context (+USE, transcripts -54/+134, lanes 1-4), the 5[prime]-context (-USE, +1/+134, lanes 5-8), or an extended 5[prime]-like transcript (-USE, +leader, +1/+368, lanes 9-12). The position of the RNA and RNA-protein complexes are indicated on the left. Part of the gel with the free, uncomplexed RNA templates is shown in the lower panel. A surprising result is that the +1/+368 HIV-1 transcript is an inactive template for binding of polyadenylation factors (Fig. To prove that the leader-mediated inhibition is sequence-specific and not due to 3[prime]-extension of the transcript, we synthesized a 389 nt control transcript in which the 5[prime] poly(A) site was extended by vector-derived sequences that are unrelated to HIV-1 (+1/+180R, see Fig. Several of the sequences that comprise the HIV-1 poly(A) site are positioned within an RNA stem-loop structure that we termed the poly(A) hairpin. Occluded by basepairing are part of the AAUAAA hexamer motif, the CA cleavage site and part of the downstream GU-rich element (Fig. Figure 7. Leader-mediated inhibition of the upstream HIV-1 poly(A) site. Two sets of wild-type HIV-1 transcripts were incubated with HeLa nuclear extract. We analyzed the 3[prime]-like transcript set (lanes 1-6) and the 5[prime]-like transcripts (lanes 7-12), both with variable 3[prime] ends (indicated on top of the lanes). Transcript -54/+202 and 3[prime]-extended versions thereof contain both the 3[prime]-specific USE enhancer and part of the 5[prime]-specific leader region, and thus represent unnatural, hybrid 5[prime]/3[prime] RNAs. The +1 set is also shown as free, uncomplexed RNA (lanes 13-18). The position of the poly(A) complex is indicated on the left, and the position of RNA monomers and RNA dimers are marked on the right. Thus, we measured an inverse correlation between the thermodynamic stability of the poly(A) hairpin and its ability to interact with polyadenylation factors. Furthermore, the efficiency of poly(A) complex formation of the different transcripts correlated with the polyadenylation activity that we measured previously in transfected cells (46). Hardly any binding was observed with hairpins with a [Delta]G value below -19.9 kcal/mol (A2 revertant, free energy was calculated at 30°C, which is the temperature of the EMSA assay). Even the wild-type hairpin ([Delta]G = -18.1 kcal/mol at 30°C) was repressive under certain experimental conditions. For instance, inefficient binding of polyadenylation factors was observed in HeLa nuclear extract with the wild-type HIV-1 transcript that mimics the 5[prime] poly(A) site (+1/+134), which lacks the USE enhancer. The ability of this wild-type transcript to form poly(A) complexes could be improved in two ways; by inclusion of the USE enhancer (transcript -54/+134) or by destabilization of the hairpin structure (mutants B and C). In other words, it appears that local RNA structure partially suppresses the activity of the HIV-1 poly(A) signal, which is the ideal situation to enable further up-regulation by the USE in the 3[prime]-context and further down-regulation by SD/leader sequences in the 5[prime] context. Thus, a complex interplay of polyadenylation and splicing signals, repressive RNA structure and enhancer/silencer elements are involved in regulated HIV-1 polyadenylation. The USE element is necessary for efficient processing at the HIV-1 poly(A) site (22-25), and was shown to act through binding of CPSF (27). We propose that the wild-type HIV-1 RNA template, which cannot optimally interact with CPSF because of local RNA structure, uses the upstream USE motif to overcome this deficiency in CPSF-binding. A striking similar observation has been made by Gilmartin et al. (27). These authors showed that the USE is necessitated by the suboptimal sequence context of the HIV-1 AAUAAA hexamer. We think that this suboptimal context represents the local RNA structure. Indeed, RNA mutants with a destabilized hairpin are not responsive to the USE enhancer, presumably because their AAUAAA motif is not occluded. It has been suggested that the presence of the USE may enable CPSF to identify the correct AAUAAA hexamer (27). We now propose a new mechanistic model for recognition of the 3[prime] HIV-1 poly(A) site that incorporates all of the above findings (Fig. Figure 8. Model of structure-mediated repression and USE-mediated activation of the 3[prime] poly(A) site of the HIV-1 genome. The key regulatory motifs that control polyadenylation are indicated. At both the 5[prime] and 3[prime] poly(A) site we have drawn the poly(A) hairpin structure that occludes the AAUAAA hexamer (black triangle). At the 5[prime] site we have indicated the downstream repressive (-) elements: the major SD (black box) and the leader sequences. At the 3[prime] poly(A) site the stimulatory (+) USE element is shown (open box), which is proposed to act as initial binding site or entry site for CPSF (grey circle). Upon transient opening or breathing of the hairpin, CPSF will bind the AAUAAA hexamer. See the text for further details. According to the proposed mechanism, HIV-1 RNA has a bipartite CPSF binding site in the 3[prime] poly(A) site (USE and AAUAAA). In contrast, only the AAUAAA motif is available in the 5[prime] poly(A) site, which will be occluded by the repressive hairpin structure that is expected to fold rapidly. For instance, early investigations into the folding of the tRNA molecule established an approximate time scale for the formation of RNA secondary structure in the 10-4 to 10-5 s range (reviewed in 65). However, the RNA structure-mediated inhibition of polyadenylation may not be sufficient for complete suppression of 5[prime] polyadenylation because the hairpin structure will be in equilibrium with the open form, and this `breathing' will eventually expose the AAUAAA signal. Rapid folding of the poly(A) hairpin on the nascent transcript will delay recognition by polyadenylation factors, such that sufficient time is available for the additional repressive mechanisms to become effective. For instance, the growing RNA chain will at a certain point be recognized by the splicing machinery, and assembly of spliceosomes may hinder the interaction with polyadenylation factors. It has been reported that binding of U1 snRNP to the major SD site in the HIV-1 leader RNA leads to suppression of the 5[prime] poly(A) site (41). Molecular details of this mechanism are currently unknown, but this example adds to the growing list of cases in which the splicing machinery influences the process of polyadenylation (14). Our RNA binding studies suggest that the leader region can also inhibit the binding of polyadenylation factors to the 5[prime] poly(A) site in a more direct manner. This negative effect is specific for the HIV-1 leader sequences, although we were unable to identify a distinct repressive element. We measured that the binding of polyadenylation factors is gradually decreased upon inclusion of additional HIV-1 sequences, and it is possible that the leader RNA will bit-by-bit adopt a higher order structure that step-by-step restricts the accessibility of the 5[prime] poly(A) site. This direct repressive effect of the leader region may add to the SD-mechanism (40,41) and suppression by promoter proximity (37,38) to strongly inhibit the 5[prime] poly(A) site. Despite these multiple repressive mechanisms, we measured that ~5-10% of the HIV-1 transcripts are prematurely polyadenylated at the 5[prime] poly(A) site in virus-infected cells (66). In conclusion, we propose that the regulated step of HIV-1 mRNA polyadenylation is the binding of CPSF to the AAUAAA sequence motif. This is mediated by sequestering of the polyadenylation signal within the poly(A) hairpin structure. In the context of the 5[prime] poly(A) site, additional repressive effects lead to nearly complete inhibition of polyadenylation. In the context of the 3[prime] poly(A) site, the USE element acts as an entry site for the CPSF factor, and thereby facilitates poly(A) complex formation. Some of the predictions of this model were tested recently in full-length HIV-1 constructs with specific mutations introduced in either the 5[prime] or 3[prime] poly(A) hairpin (66). Opening of the 5[prime] hairpin activated premature polyadenylation up to 30-40% efficiency, causing a concomitant decline in viral RNA synthesis. This result demonstrates that this RNA structure is pivotal for repression of the 5[prime] poly(A) site. Whereas the wild-type hairpin obviously does not interfere with efficient polyadenylation at the 3[prime] poly(A) site, this mRNA processing step is blocked by the stabilized hairpin mutant. Thus, the thermodynamic stability of this RNA structure is delicately balanced to allow efficient repression of the 5[prime] poly(A) site, yet full activity of the 3[prime] poly(A) site. This may explain the apparent conservation of this structured RNA motif and its thermodynamic stability among all human and simian immunodeficiency viruses (42). It is likely that the proposed mechanism of regulated polyadenylation represents a more general gene expression strategy of the complex retroviruses. A common mechanism has been proposed for the equine infectious anemia virus (EIAV), which is a related lentivirus (26). Furthermore, we reported that both the lentivirus and spumavirus groups can fold a similar RNA structure that occludes part of the poly(A) signal (66). The human T-cell leukemia virus (HTLV-I), a representative of the oncoretroviruses, also uses RNA secondary structure as a regulatory element in the process of polyadenylation (67-69). A complex RNA structure is formed at the 3[prime] end of the viral genome that juxtaposes the AAUAAA hexamer and the cleavage site, which are separated by 274 nucleotides in the linear sequence. These examples underscore the versatile usage by retroviruses of RNA structure as a key component of regulatory circuits. We thank Maarten de Smit for advice and critical reading of the manuscript and Wim van Est for artwork. This work was supported in part by the Dutch AIDS Fund (AIDS Fonds) and the EC (grant 950675).
MATERIALS AND METHODS
Plasmid constructs
In vitro transcription reactions
Preparation of nuclear extracts and purified CPSF/CstF
EMSA
RESULTS
Stable RNA structure interferes with the binding of polyadenylation factors
Revertants of the stabilized mutant hairpin rescue binding of polyadenylation factors
The USE enhancer specifically overcomes the structure-mediated repression of the HIV-1 poly(A) site
Leader-specific inhibition of the HIV-1 poly(A) site
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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