Design of RNA-binding proteins and ligands.

The rapidly expanding database of RNA structures and protein complexes is beginning to lead to the successful design of specific RNA-binding molecules. Recent combinatorial and structure-based approaches have utilized known nucleic-acid-binding scaffolds from both proteins and small molecules to display a relatively small set of functional groups often used in protein--RNA recognition. Several studies have shown that the tethering of multiple binding modules can enhance RNA-binding affinity and specificity, a strategy also commonly used in DNA recognition.

Recognition of RNA branch point sequences by the KH domain of splicing factor 1 (mammalian branch point binding protein) in a splicing factor complex.

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a "maxi-K homology" (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, we constructed a transcriptional reporter system utilizing human immunodeficiency virus type 1 Tat fusion proteins and examined the RNA-binding specificity of the complex using KH domain and RNA-binding site mutants. We first established that SF1/mBBP and U2AF cooperatively assemble in our reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. We next found that the activities of the Tat fusion proteins on different BPS variants correlated well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by "simple" KH domains, involving residues from two putative alpha helices, a highly conserved loop, and parts of a beta sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex (Lewis et al., Cell 100:323-332, 2000), we propose a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites.

Molecular dynamics and binding specificity analysis of the bovine immunodeficiency virus BIV Tat-TAR complex.

We have performed molecular dynamics (MD) simulations, with particle-mesh Ewald, explicit waters, and counterions, and binding specificity analyses using combined molecular mechanics and continuum solvent (MM-PBSA) on the bovine immunodeficiency virus (BIV) Tat peptide-TAR RNA complex. The solution structure for the complex was solved independently by Patel and co-workers and Puglisi and co-workers. We investigated the differences in both structures and trajectories, particularly in the formation of the U-A-U base triple, the dynamic flexibility of the Tat peptide, and the interactions at the binding interface. We observed a decrease in RMSD in comparing the final average RNA structures and initial RNA structures of both trajectories, which suggests the convergence of the RNA structures to a MD equilibrated RNA structure. We also calculated the relative binding of different Tat peptide mutants to TAR RNA and found qualitative agreement with experimental studies.

Structural characterization of the complex of the Rev response element RNA with a selected peptide.

INTRODUCTION: The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. RESULTS: The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine-purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. CONCLUSIONS: These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.

Structure-based design of a dimeric RNA-peptide complex.

The arginine-rich RNA-binding domain of bovine immunodeficiency virus (BIV) Tat adopts a beta-hairpin conformation upon binding to the major groove of BIV TAR. Based on its NMR structure, we modeled dimeric arrangements in which two adjacent TAR sites might be recognized with high affinity by a dimeric peptide. Some dimeric RNAs efficiently bound two unlinked BIV Tat peptides in vitro, but could not bind even one monomeric peptide in vivo, as monitored by transcriptional activation of human immunodeficiency virus long terminal repeat reporters. Results with additional reporters suggest that extending the RNA helix in the dimeric arrangements inhibits peptide binding by decreasing major groove accessibility. In contrast, a dimeric peptide efficiently bound an optimally arranged dimeric TAR in vivo, and bound with an affinity at least 10-fold higher than the monomeric peptide in vitro. Mutating specific nucleotides in each RNA 'half-site' or specific amino acids in each beta-hairpin of the dimeric peptide substantially decreased binding affinity, providing evidence for the modeled dimer-dimer interaction. These studies provide a starting point for identifying dimeric RNA-protein interactions with even higher binding affinities and specificities.

An RNA-binding chameleon.

The arginine-rich RNA binding motif is found in a wide variety of proteins, including several viral regulatory proteins. Although related at the primary sequence level, arginine-rich domains from different proteins adopt different conformations depending on the RNA site recognized, and in some cases fold only in the context of RNA. Here we show that the RNA binding domain of the Jembrana disease virus (JDV) Tat protein is able to recognize two different TAR RNA sites, from human and bovine immunodeficiency viruses (HIV and BIV, respectively), adopting different conformations in the two RNA contexts and using different amino acids for recognition. In addition to the conformational differences, the JDV domain requires the cyclin T1 protein for high-affinity binding to HIV TAR, but not to BIV TAR. The "chameleon-like" behavior of the JDV Tat RNA binding domain reinforces the concept that RNA molecules can provide structural scaffolds for protein folding, and suggests mechanisms for evolving distinct RNA binding specificities from a single multifunctional domain.

Fitting peptides into the RNA world.

The structures of several peptide-RNA complexes have been reported in the past year, underscoring the diverse nature of RNA structure and protein interactions. In general, specific peptide conformations are stabilized by the surrounding RNA framework; this is strikingly similar to how peptides are stabilized upon interaction with proteins.

Structure determination and binding kinetics of a DNA aptamer-argininamide complex.

The structure of a DNA aptamer, which was selected for specific binding to arginine, was determined using NMR spectroscopy. The sequence forms a hairpin loop, with residues important for binding occurring in the loop region. Binding of argininamide induces formation of one Watson-Crick and two non-Watson-Crick base pairs, which facilitate generation of a binding pocket. The specificity for arginine seems to arise from contacts between the guanidino end of the arginine and phosphates, with atoms positioned by the shape of the pocket. Complex binding kinetics are observed suggesting that there is a slow interconversion of two forms of the DNA, which have different binding affinities. These data provide information on the process of adaptive recognition of a ligand by an aptamer.

Screening in vivo for RNA-binding peptides from combinatorial libraries.

We have modified a previously developed genetic assay system for RNA-polypeptide interactions in a attempt to more readily identify RNA-binding peptides. The first modification involved the design of a "complex" library that would contain a variety of RNA-binding polypeptides. The second modification involved the use of neomycin phosphotransferase (NPT II) as the reporter gene, therefore allowing "selection" of RNA-binding peptides by kanamycin resistance. The improved screening system should allow the identification of peptides that bind to a variety of RNA structures.

If the loop fits...

The structure of the yeast L30 ribosomal protein bound to its autoregulatory RNA site has been determined by NMR spectroscopy. The intricate architecture of the RNA internal loop and the structure of the binding region of the protein both are stabilized in the complex, highlighting the importance of mutually-induced fit in RNA-protein interactions.

Structure-based design of an RNA-binding zinc finger.

A structure-based approach was used to design RNA-binding zinc fingers that recognize the HIV-1 Rev response element (RRE). An arginine-rich alpha-helix from HIV-1 Rev was engineered into the zinc finger framework, and the designed fingers were shown to bind specifically to the RRE with high affinity and in a zinc-dependent manner, and display cobalt absorption and CD spectra characteristic of properly folded fingers. The results indicate that a monomeric zinc finger can recognize a specific nucleic acid site and that the alpha-helix of a finger can be used to recognize the major groove of RNA as well as DNA. The RRE-binding zinc fingers demonstrate how structure-based approaches may be used in the design of potential RNA-binding therapeutics and provide a framework for selecting RNA-binding fingers with desired specifications.

In vivo selection of RNA-binding peptides from combinatorial libraries.

We have used a two step procedure to identify peptides that bind strongly to the Rev-response element (RRE) of HIV. In the first step, RRE-binding peptides were screened from a combinatorial peptide library generated by "randomization" using a small subset of the 20 amino acids. In the second step, one such RRE-binding peptide, RSG-1, was "evolved" into an even stronger RRE-binding peptide using a codon-based mutagenesis procedure. After 2 rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity than wild-type Rev peptide.

HIV-1: fifteen proteins and an RNA.

Human immunodeficiency virus type 1 is a complex retrovirus encoding 15 distinct proteins. Substantial progress has been made toward understanding the function of each protein, and three-dimensional structures of many components, including portions of the RNA genome, have been determined. This review describes the function of each component in the context of the viral life cycle: the Gag and Env structural proteins MA (matrix), CA (capsid), NC (nucleocapsid), p6, SU (surface), and TM (transmembrane); the Pol enzymes PR (protease), RT (reverse transcriptase), and IN (integrase); the gene regulatory proteins Tat and Rev; and the accessory proteins Nef, Vif, Vpr, and Vpu. The review highlights recent biochemical and structural studies that help clarify the mechanisms of viral assembly, infection, and replication.

Altering the context of an RNA bulge switches the binding specificities of two viral Tat proteins.

The bovine immunodeficiency virus (BIV) Tat protein binds with high affinity to its TAR RNA site through a large set of specific RNA-protein contacts whereas human immunodeficiency virus (HIV) Tat makes relatively few contacts to HIV TAR and requires the assistance of a cellular protein to bind with high affinity. The two TAR sites are structurally very similar, but BIV Tat appears unable to make the same set of high-affinity contacts to HIV TAR. To determine the basis of this discrimination, we examined BIV Tat binding to a series of hybrid TARs both in vivo and in vitro. We expected that differences in the architectures of the bulges might account for the binding specificity; however, the results show that flanking base pairs provide the key determinants. Based on these data, we designed a novel TAR that is recognized by both BIV Tat and HIV Tat. This RNA may be viewed as a primordial TAR from which two distinct recognition strategies can be evolved.

A novel glutamine-RNA interaction identified by screening libraries in mammalian cells.

The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These results, together with the structure of a Rev peptide-Rev response element complex, suggest that the carboxamide groups of glutamine or asparagine are well-suited to hydrogen bond to G-A base pairs and begin to establish an RNA recognition code for the arginine-rich motif. The screening approach may provide a relatively general method for screening expression libraries in mammalian cells.