Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

RNA-binding activities of the different domains of a spinach chloroplast ribonucleoprotein.

An RNA-binding protein of 28 kD (28RNP) has been previously isolated from spinach chloroplasts and was found to be required for 3' end processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic and glycine-rich amino terminal domain. Each domain by itself, as well as in combination with other domains, was expressed in bacterial cells and the polypeptides were purified to homogeneity. We have investigated the RNA-binding properties of the different structural domains using UV-crosslinking, saturation binding and competition between the different domains on RNA-binding. It was found that the acidic domain does not bind RNA, but that each of the RNA-binding domains, expressed either individually or together, do bind RNA, although with differing affinities. When either the first or second RNA-binding domain was coupled to the acidic domain, the affinity for RNA was greatly reduced. However, the acidic domain has a positive effect on the binding of the full-length protein to RNA, because the mature protein binds RNA with a better affinity than the truncated protein which lacks the acidic domain. In addition, it was found that a stretch of two or three G residues is enough to mediate binding of the 28RNP, whereas four U residues were insufficient. The implications of the RNA-binding properties of 28RNP to its possible function in the processing of chloroplast RNA is discussed.     

Design and development of a catalytic ribonucleoprotein

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.     

Ribonucleopeptides: functional RNA-peptide complexes

Selection of functional RNAs from randomized pool of RNA molecules successfully affords RNA aptamers that specifically bind to small molecules, and that have catalytic activities. Recent structural analyses of the ribosomal RNA complex suggest that the RNA-protein complex would be a new structural candidate for the design of tailor-made receptors and enzymes. We have designed an ATP binding domain that consists of an RNA subunit and a peptide subunit by means of structure-based design approach and successive in vitro selection method. The RNA subunit is designed to consist of two functional domains; an ATP binding domain with 20 randomized nucleotides and an adjacent stem region that serves as a binding site for the RNA-binding peptide. The randomized nucleotide region was placed next to the HIV-1 Rev response element to enable the formation of "ribonucleopeptide" pools in the presence of the Rev peptide. In vitro selection of RNA oligonucleotides from the randomized pool afforded a ribonucleopeptide receptor specific for ATP. The ATP-binding ribonucleopeptide did not share the known consensus nucleotide sequence for ATP aptamers, and completely lost its ATP-binding ability in the absence of the Rev peptide. The ATP-binding activity of the ribonucleopeptide was increased by a substitution of the N-terminal amino acid of the Rev peptide. These results demonstrate that the peptide stabilizes the functional structure of RNA and suggest that amino acids outside the RNA binding region of the peptide participate in the ATP binding. Our approach would provide a new strategy for the design of tailor-made ribonucleopeptide receptors.     

HIV Rev peptides conjugated with peptide nucleic acids and their efficient binding to RRE RNA

HIV Rev peptides conjugated with peptide nucleic acids (PNAs) were designed and synthesized to develop a designing approach for a novel RNA-binding molecule. The binding affinities of PNA-peptides with the Rev responsive element (RRE) RNA were determined by the competition assay using a rhodamine-labeled Rev. The peptide conjugated with an antisense PNA (TGCGC) bound RRE RNA more efficiently than the molecule without the PNA or the peptide sequence.     

Evolvability of the mode of peptide binding by an RNA

The HIV Rev-response element (RRE) RNA binds strongly to two unrelated peptides, the HIV Rev peptide and an RRE-binding aptamer, the RSG-1.2 peptide, at a similar site, but using distinct sets of interactions. In this study, the nucleotide base requirements for the binding of the RRE to the Rev and RSG-1.2 peptides were determined by selection of Rev- and RSG-1.2-binding RRE variants using a bacterial reporter system. As a result, distinct differences in the bases necessary for binding the two peptides were found in the upper stem of the RRE. Strikingly, single nucleotide changes in this region were found to switch the peptide-binding specificity of the RRE from a bifunctional Rev- and RSG-1.2-binding mode to either a Rev-specific or a RSG-1.2- specific mode, demonstrating how an RNA can evolve alternative binding strategies in discrete steps without intermediate loss of function. This evolvability of the mode of peptide binding by an RNA presumably reflects the multidimensionality of conformational space that a given RNA has available for ligand recognition, which may have been utilized in the evolution of RNA-polypeptide complexes.     

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.     

Assignment and modeling of the Rev Response Element RNA bound to a Rev peptide using 13C-heteronuclear NMR

Summary The Rev Response Element (RRE) RNA-Rev protein interaction is important for regulation of gene expression in the human immunodeficiency virus. A model system for this interaction, which includes stem IIB of the RRE RNA and an arginine-rich peptide from the RNA-binding domain of Rev, was studied using multidimensional heteronuclear NMR. Assignment of the RNA when bound to the peptide was obtained from NMR experiments utilizing uniformly and specifically ¹³C-labeled RNA. Isotopic filtering experiments on the specifically labeled RNA enabled unambiguous assignment of unusual nonsequential NOE patterns present in the internal loop of the RRE. A three-dimensional model of the RNA in the complex was obtained using restrained molecular dynamics calculations. The internal loop contains two purine-purine base pairs, which are stacked to form one continuous helix flanked by two A-form regions. The formation of a G-G base pair in the internal loop requires an unusual structure of the phosphate backbone. This structural feature is consistent with mutational data as being important for the binding of Rev to the RRE. The G-G base pair may play an important role in opening the normally narrow major groove of A-form RNA to permit binding of the Rev basic domain.     

A case of convergent evolution of nucleic acid binding modules

Divergent evolution can explain how many proteins containing structurally similar domains, which perform a variety of related functions, have evolved from a relatively small number of modules or protein domains. However, it cannot explain how protein domains with similar, but distinguishable, functions and similar, but distinguishable, structures have evolved. Examples of this are the RNA-binding proteins containing the RNA-binding domain (RBD), and a newly established protein group, the cold-shock domain (CSD) protein family. Both protein domains contain conserved RNP motifs on similar single-stranded nucleic acid-binding surfaces. Apart from the RNP motifs, which have a similar function, the two families show little similarity in topology or amino acid sequence. This can be considered an interesting example of convergent evolution at the molecular level. Previously, a β-sheet surface was found to interact with RNA in non-homologous proteins from yeast, phage and man, revealing that this mode of RNA binding may be a widely recurring theme.     

Sensitive in vitro analysis of HIV-1 Rev multimerization

Oligomerization of the Rev protein of human immuno-deficiency virus type 1 on its cognate response element is essential for export of the late viral mRNAs from the nucleus. Two regions of the protein, flanking the RNA binding site, have been defined as oligomerization sites after mutants (M4 and M7) had been reported to bind specifically to the response element but not to oligomerize in vivo or in vitro. These mutants are often used as paradigms for studies of Rev multimerization. We have re-examined the in vitro binding of these mutants to model Rev response elements, using improved gel mobility assays. We find that both mutants will form oligomers on the Rev response element, but have somewhat lower affinities for RNA than the wild-type protein. M7 has lower specific affinity, but shows little deficiency in oligomerization once binding starts. In contrast, M4 is multimerization deficient, as previously reported. Therefore, whilethe sites are correctly defined, it is inappropriate to employ the original M7 deletion mutant to study Rev oligomerization.     

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.     

Real-time kinetics of HIV-1 Rev-Rev response element interactions. Definition of minimal binding sites on RNA and protein and stoichiometric analysis.

The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10(-10) M, whereas the affinity for the core RNA was about 10(-11) M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.     

Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA.

An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain. Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs. It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs. In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases. It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with [32P] orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II. The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein. Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan. RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein. Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast.     

Mechanism of neomycin and Rev peptide binding to the Rev responsive element of HIV-1 as determined by fluorescence and NMR spectroscopy

Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short alpha-helical peptide derived from Rev (Rev 34-50), and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2'-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K(D) = 20 +/- 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K(D) = 0.24 +/- 0.040 microM), the second site inhibited Rev binding in a competitive fashion (K(D) = 1. 8 +/- 0.8 microM), and the third much weaker site (or sites) is attributed to nonspecific binding (K(D) >/= 40 microM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE.     

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.     

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.     

The Cbf5-Nop10 complex is a molecular bracket that organizes box H/ACA RNPs

Box H/ACA ribonucleoprotein particles (RNPs) catalyze RNA pseudouridylation and direct processing of ribosomal RNA, and are essential architectural components of vertebrate telomerases. H/ACA RNPs comprise four proteins and a multihelical RNA. Two proteins, Cbf5 and Nop10, suffice for basal enzymatic activity in an archaeal in vitro system. We now report their cocrystal structure at 1.95-A resolution. We find that archaeal Cbf5 can assemble with yeast Nop10 and with human telomerase RNA, consistent with the high sequence identity of the RNP components between archaea and eukarya. Thus, the Cbf5-Nop10 architecture is phylogenetically conserved. The structure shows how Nop10 buttresses the active site of Cbf5, and it reveals two basic troughs that bidirectionally extend the active site cleft. Mutagenesis results implicate an adjacent basic patch in RNA binding. This tripartite RNA-binding surface may function as a molecular bracket that organizes the multihelical H/ACA and telomerase RNAs.