High-resolution mapping of the human T-cell leukemia virus type 1 Rex-binding element by in vitro selection.

Interactions between the Rex protein of HTLV-1 and the genomic Rex-binding element (XBE) mediate the cytoplasmic transport of viral mRNAs. However, it is uncertain which RNA sequences and structures contribute to Rex recognition. A portion of the viral genome that spanned the XBE was partially randomized, and functional Rex-binding variants were selected. Alignment of selected Rex-binding sequences revealed positions that were functionally conserved between different molecules. A model is presented in which a subset of the selected residues are in direct contact with Rex. Positions that covaried with one another were also found. These covariations support a secondary-structural model in which a central paired stem is symmetrically flanked by two bulge loops. On the basis of this model, site-directed mutations of the XBE were constructed and each half molecule was found to bind independently to Rex. The functional residues and secondary structures in the XBE half molecules bear a remarkable resemblance to the transactivation response region element of HIV-1. Since the transactivation response region element is known to interact specifically with arginine residues in the Tat protein, these results suggest that the XBE binds to the arginine-rich RNA-binding domain of Rex in a similar manner. This model is supported by the selection data.     

RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.     

The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner

Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5′ terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5′-GUAAC / 3′-CUUAG double-stranded motif, which closely resembles the canonical 5′-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar K_D values) that leads to oligomerization of the RBD on its RNA ligands.     

Posttranscriptional regulation by the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins through a heterologous RNA binding site.

The human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex proteins induce cytoplasmic expression of incompletely spliced viral mRNAs by binding to these mRNAs in the nucleus. Each protein binds a specific cis-acting element in its target RNAs. Both proteins also associated with nucleoli, but the significance of this association is uncertain because mutations that inactivate nucleolar localization signals in Rev or Rex also prevent RNA binding. Here we demonstrate that Rev and Rex can function when tethered to a heterologous RNA binding site by a bacteriophage protein. Under these conditions, cytoplasmic accumulation of unspliced RNA occurs without the viral response elements, mutations in the RNA binding domain of Rev do not inhibit function, and nucleolar localization can be shown to be unnecessary for the biological response.     

Uptake and intracellular transport of RNA aptamers in African trypanosomes suggest therapeutic "piggy-back" approach

African trypanosomes are protozoan organisms that multiply as extracellular parasites in the blood of humans and other mammals. The parasites escape destruction by the host immune system by periodically changing their glycoprotein surface coat. This phenomenon is known as antigenic variation and is responsible for the inability of the infected host to clear the infection. Previously we reported the selection of RNA aptamers that bind to a 42 kDa surface protein of Trypanosoma brucei. The polypeptide is localised within a specific substructure on the parasite surface, the so-called flagellar pocket. Here we analyse the fate of the aptamers upon binding to the flagellar pocket. At elevated temperatures, both terminal ends of the RNAs are degraded to form a stable core structure of approximately 50 nucleotides. The RNAs become rapidly internalised by endocytosis and are transported to the lysosome by vesicular transport. The endocytotic process is sequence specific and does not occur with randomised RNA sequences or significantly shortened aptamer fragments. Co-localisation experiments with transferrin suggest a receptor-mediated uptake. The identified internalisation and transport pathway was used to target aptamer-coupled biotin molecules to the lysosome. This demonstrates that the RNAs can be used as 'piggy-back' molecules to target aptamer-coupled compounds/toxins to the lysosomal compartment of the parasite.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

Hepatitis C virus (HCV)-encoded nonstructural protein 3 (NS3) possesses protease, NTPase, and helicase activities, which are considered essential for viral proliferation. Thus, HCV NS3 is a good putative therapeutic target protein for the development of anti-HCV agents. In this study, we isolated specific RNA aptamers to the helicase domain of HCV NS3 from a combinatorial RNA library with 40-nucleotide random sequences using in vitro selection techniques. The isolated RNAs were observed to very avidly bind the HCV helicase with an apparent Kd of 990 pM in contrast to original pool RNAs with a Kd of >1 microM. These RNA ligands appear to impede binding of substrate RNA to the HCV helicase and can act as potent decoys to competitively inhibit helicase activity with high efficiency compared with poly(U) or tRNA. The minimal binding domain of the ligands was determined to evaluate the structural features of the isolated RNA molecules. Interestingly, part of binding motif of the RNA aptamers consists of similar secondary structure to the 3'-end of HCV negative-strand RNA. Moreover, intracellular NS3 protein can be specifically detected in situ with the RNA aptamers, indicating that the selected RNAs are very specific to the HCV NS3 helicase. Furthermore, the RNA aptamers partially inhibited RNA synthesis of HCV subgenomic replicon in Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV helicase mechanism.     

Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved “GGGUGG” and “GAGGG” motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP’s antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.     

A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.     

Examination of specific binding activity of aptamer RNAs to the HIV-NC by using a cell-based in vivo assay for protein-RNA interaction

The nucleocapsid (NC) protein of the Human Immunodeficiency Virus-1 plays a key role in viral genomic packaging by specifically recognizing the Psi(Psi) RNA sequence within the HIV-1 genome RNA. Recently, a novel cell-based assay was developed to probe the specific interactions in vivo between the NC and Psi-RNA using E.coli cells (J. Virol. 81: 6151-55, 2007). In order to examine the extendibility of this cell-based assay to RNAs other than Psi-RNA, this study tested the RNA aptamers isolated in vitro using the SELEX method, but whose specific binding ability to NC in a living cellular environment has not been established. The results demonstrate for the first time that each of those aptamer RNAs can bind specifically to NC in a NC zinc finger motif dependent manner within the cell. This confirms that the cell-based assay developed for NC-Psi interaction can be further extended and applied to NC-binding RNAs other than Psi-RNA.     

RNA apatamers for yeast ribosomal protein L32 have a conserved purine-rich internal loop.

Two in vitro selection experiments were conducted to determine the RNA sequence requirements for binding ribosomal protein L32 (RPL32) from Saccharomyces cerevisiae. To preserve the wild-type stem-internal loop-stem fold, only a limited portion of the RNA comprising the internal loop region was randomized. Most of the selected RNAs have secondary structures similar to that of the wild-type, and four purines on both sides of the internal loop are highly conserved. Indeed, a pair of 5'-GA-3' dinucleotides is found in all but one of the stem-loop-stem L32 aptamers and these conserved purines may contact the protein directly or form a necessary RNA secondary or tertiary structure. These aptamers have a potential G:U pair bordering the loop adjacent to the conserved GAs, but a cytidine replaces a phylogenetically conserved adenosine at one loop position in many of the selected RNAs. In model RNAs, the cytidine-bearing variant binds protein slightly more strongly than does the wild-type RNA. That the seven-member, 2 + 5 internal loop is important for protein binding is reinforced by the finding that the position, but not the size, of the loop is variable. A minority of the RNA aptamers has three consecutive uridines and may fold into a similar structure, but with the internal loop inverted.     

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.     

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.     

Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation

Although nascent noncoding RNAs can undergo maturation to functional RNAs or degradation by quality control pathways, the events that influence the choice of pathway are not understood. We report that the targeting of pre-tRNAs and certain other noncoding RNAs for decay by the TRAMP pathway is strongly influenced by competition between the La protein and the Rex1 exonuclease for access to their 3' ends. The La protein binds the 3' ends of many nascent noncoding RNAs, protecting them from exonucleases. We demonstrate that unspliced, end-matured, partially aminoacylated pre-tRNAs accumulate in yeast lacking the TRAMP subunit Trf4p, indicating that these pre-tRNAs normally undergo decay. By comparing RNA extracted from wild-type and mutant yeast strains, we show that Rex1p is the major exonuclease involved in pre-tRNA trailer trimming and may also function in nuclear CCA turnover. As the accumulation of end-matured pre-tRNAs in trf4Delta cells requires Rex1p, these pre-tRNAs are formed by exonucleolytic trimming. Accumulation of truncated forms of 5S rRNA and SRP RNA in trf4Delta cells also requires Rex1p. Overexpression of the La protein Lhp1p reduces both exonucleolytic pre-tRNA trimming in wild-type cells and the accumulation of defective RNAs in trf4Delta cells. Our experiments reveal that one consequence of Rex1p-dependent 3' trimming is the generation of aberrant RNAs that are targeted for decay by TRAMP.     

Recombination, RNA evolution, and bifunctional RNA molecules isolated through chimeric SELEX.

Exchange of RNA structural domains through recombination can be used to engineer RNAs with novel functions and may have played an important role in the early evolution of life. The degree of function an RNA element retains upon recombination into a new sequence context is a measure of how deleterious or beneficial recombination will be. When we fused pairs of aptamers previously selected to bind coenzyme A, chloramphenicol, or adenosine, the chimerae retained some ability to bind both targets, but with reduced binding activity both in solution and on affinity resins, probably due to misfolding. Complex populations of recombined RNAs gave similar results. Applying dual selection pressure to recombined populations yielded the combinations that were best suited to binding both targets. Most reselected RNAs folded into the active conformation more readily than chimerae built from arbitrarily chosen aptamers, as indicated both by solution Kd measurements and affinity resin binding activity. Deletion/selection experiments confirmed that the sequences required for binding are fully contained within the respective domains and not derived from interaction between the domains, consistent with the modular architecture of their original design. The combinatorial nature of the recombination methods presented here takes advantage of the full sequence diversity of the starting populations and yields large numbers of bifunctional molecules (10(6) to more than 1012). The method can be easily generalized and should be applicable to engineering dual-function RNAs for a wide variety of applications, including catalysis, novel therapeutics, and studies of long-range RNA structure.