Gbp1p, a Protein with RNA Recognition Motifs, Binds Single-Stranded Telomeric DNA and Changes Its Binding Specificity upon Dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site

Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

Analysis of nucleic acid binding by a recombinant translin-trax complex

Translin is a highly conserved mammalian RNA and DNA-binding protein involved in DNA recombination and RNA trafficking. Crystal structures of mouse and human translin have been solved, but do not provide information about nucleic acid binding or recognition. Translin has a partner protein, translin-associated factor x (trax), which is believed to regulate translin’s subcellular locale and affinity for certain RNA and DNA sequences. Here we present a comparative study of recombinant translin and translin-trax complex binding to specific RNA and DNA sequences. It was observed that translin preferentially binds to G-rich RNA sequences whereas translin-trax preferentially binds G-rich DNA sequences. Translin can bind mRNA sequences with sub-micromolar K_d values, and the complex with trax can bind G-rich DNA with similar affinity. We conclude that trax acts to regulate translin’s RNA and DNA binding affinities as part of a cellular RNA trafficking mechanism.     

Identification of a 14mer RNA that recognizes and binds flavin mononucleotide with high affinity

Aptamers are nucleic acids developed by in vitro evolution techniques that bind to specific ligands with high affinity and selectivity. Despite such high affinity and selectivity, however, in vitro evolution does not necessarily reveal the minimum structure of the nucleic acid required for selective ligand binding. Here, we show that a 35mer RNA aptamer for the cofactor flavin mononucleotide (FMN) identified by in vitro evolution can be computationally evolved to a mere 14mer structure containing the original binding pocket and eight scaffolding nucleotides while maintaining its ability to bind in vitro selectively to FMN. Using experimental and computational methodologies, we found that the 14mer binds with higher affinity to FMN (K_D ~ 4 µM) than to flavin adenine dinucleotide (K_D ~ 12 µM) or to riboflavin (K_D ~ 13 µM),despite the negative charge of FMN. Different hydrogen-bond strengths resulting from differing ring-system electron densities associated with the aliphatic-chain charges appear to contribute to the selectivity observed for the binding of the 14mer to FMN and riboflavin. Our results suggest that high affinity and selectivity in ligand binding is not restricted to large RNAs, but can also be a property of extraordinarily short RNAs.     

Nucleic acid binding properties of Escherichia coli ribosomal protein S1. I. Structure and interactions of binding site I.

Escherichia coli ribosomal protein S1 plays a central role in initiation of protein synthesis, perhaps via participation in the binding of messenger RNA to the ribosome. S1 protein has two nucleic acid binding sites with very different properties: site I binds either single-stranded DNA or RNA, while site II binds single-stranded RNA only (Draper et al., 1977). The nucleic acid binding properties of these sites have been explored using the quenching of intrinsic protein fluorescence which results from binding of oligo- and polynucleotides, and are reported in this and the accompanying paper (Draper &; von Hippel, 1978).Site I has been studied primarily using DNA oligomers and polymers, and has been found to have the following properties. (1) The intrinsic binding constant (K) of site I for poly(dA) and poly(dC) is ~3 × 10⁶m^?1 at 0.12 m-Na⁺, and the site size (n, the number of nucleotide residues covered per S1 bound) is 5.1 ± 1.0 residues. (2) Binding of site I to polynucleotides is non-co-operative. (3) The K value for binding of S1 to single-stranded polynucleotides is ~10³ larger than K for binding to double-stranded polynucleotides, meaning that S1 (via site I) is a potential “melting” or “double-helix destabilizing” protein. (4) The dependence of log K on log [Na⁺] is linear, and analysis of the data according to Record et al. (1976) shows that two basic residues in site I form charge-charge interactions with two DNA phosphates. In addition, a major part of the binding free energy of site I with the nucleic acid chain appears to involve non-electrostatic interactions. (5) Oligonucleotides bound in site II somewhat weaken the binding affinity of site I. (6) Binding affin is virtually independent of base and sugar composition of the nucleic acid ligand; in fact, the total absence of the base appears to have little effect on the binding, since the association constant for 2′-deoxyribose 5′-phosphate is approximately the same as that for dAMP or dCMP. (7) Two molecules of d(ApA) can bind to site I, suggesting the presence of two “subsites” within site I. (8) Iodide quenching experiments with S1-oligonucleotide complexes show differential exposure of tryptophans in and near the subsites of site I, depending upon whether neither, one, or both subsites are complexed with an oligonucleotide.     

Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe

Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin–TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin–TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein–protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.     

Structural Requirements for the Procoagulant Activity of Nucleic Acids

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.     

Molecular basis for the wide range of affinity found in Csr/Rsm protein–RNA recognition

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, K_d). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein–RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.     

Design and analysis of RNA structure-specific agents as potential antivirals

A number of pathogenic RNA viruses, such as HIV-1, have extensive folded RNA conformations with imperfect A-form duplexes that are essential for virus function, and could serve as targets for structure-specific antiviral drugs. A method for the discovery of such drugs involves evaluation of the interactions with RNA of a wide variety of compounds that are known to bind to nucleic acids by different mechanisms. This approach has been initiated by using corresponding sequence RNA and DNA polymers as initial test systems for analysis of RNA binding strength and selectivity. Compounds that bind exclusively in the minor groove in AT sequences of DNA do not have significant interactions with RNA. Polycations, however, can show significant RNA affinity and binding selectivity, probably through complex formation in the RNA major groove. Some intercalators and a group of diphenylfuran cations have strong interactions with RNA that are very dependent on compound structure. RNA hairpin model systems for the RRE binding site of HIV-1 Rev protein were constructed for more detailed investigations. The diphenylfuran cations bind strongly to RRE and selectively inhibit Rev binding. CD, NMR, and fluorescence binding studies indicate that the active compounds bind in the internal loop region of RRE (with binding constants >10⁷M⁻¹), and cause a conformational change in the RNA. None of the standard nucleic acid binding modes appears to fit the results for complexes of the active compounds with RRE, and it is proposed that the diphenylfuran system threads through the internal loop region of RRE. Such a model allows contacts of the furan cationic substituents with both grooves of RRE in addition to the intercalation interactions with the bases.     

Sequence-specific binding of single-stranded RNA: is there a code for recognition?

A code predicting the RNA sequence that will be bound by a certain protein based on its amino acid sequence or its structure would provide a useful tool for the design of RNA binders with desired sequence-specificity. Such de novo designed RNA binders could be of extraordinary use in both medical and basic research applications. Furthermore, a code could help to predict the cellular functions of RNA-binding proteins that have not yet been extensively studied. A comparative analysis of Pumilio homology domains, zinc-containing RNA binders, hnRNP K homology domains and RNA recognition motifs is performed in this review. Based on this, a set of binding rules is proposed that hints towards a code for RNA recognition by these domains. Furthermore, we discuss the intermolecular interactions that are important for RNA binding and summarize their importance in providing affinity and specificity.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

Binding of herpes simplex virus-1 US11 to specific RNA sequences

Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11–RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10¹⁴ 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and ≤46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.     

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and øX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m-Na⁺ or 0·1 m-Na⁺. An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono-N-ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and øX174 viral DNA with a stoichiometry of one N-ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.     

Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1

CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)_xUGU where x = 1–7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (K_d ~ 0.4 µM) is evident for RNA sequences with x = 2–4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites.     

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (T_m) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the T_m of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a T_m around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.     

A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD⁺ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.