A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L

Ribonuclease L (RNase L) is a latent endoribonuclease in an evolutionarily ancient interferon-regulated dsRNA-activated antiviral pathway. 2'-5' oligoadenylate (2-5A), the product of dsRNA-activated oligoadenylate synthetases (OASes), binds to ankyrin repeats near the amino terminus of RNase L, initiating a series of conformational changes that result in the activation of the endoribonuclease. A phylogenetically conserved RNA structure within group C enteroviruses inhibits the endoribonuclease activity of RNase L. In this study we report the mechanism by which group C enterovirus RNA inhibits RNase L. Viral RNA did not affect 2-5A binding to RNase L. Rather, the viral RNA inhibited the endoribonuclease domain. We used purified RNase L, purified 2-5A, and an RNA substrate with a 5' fluorophore and 3' quencher in FRET assays to measure inhibition of RNase L activity by the viral RNA. The group C enterovirus RNA was a competitive inhibitor of the endoribonuclease with a K(i) of 34 nM. Consistent with the kinetic profile of a competitive inhibitor, the viral RNA inhibited the constitutively active endoribonuclease domain of RNase L. We call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA).     

A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex

The yeast Saccharomyces cerevisiae ribosomal protein L30 negatively autoregulates its production by binding to a helix-loop-helix structure formed in its pre-mRNA and its mRNA. A three-dimensional solution structure of the L30 protein in complex with its regulatory RNA has been solved using NMR spectroscopy. In the complex, the helix-loop-helix RNA adopts a sharply bent conformation at the internal loop region. Unusual RNA features include a purine stack, a reverse Hoogsteen base pair (G11anti-G56syn) and highly distorted backbones. The L30 protein is folded in a three-layer alpha/beta/alpha sandwich topology, and three loops at one end of the sandwich make base-specific contacts with the RNA internal loop. The protein-RNA binding interface is divided into two clusters, including hydrophobic and aromatic stacking interactions centering around G56, and base-specific hydrogen-bonding contacts to A57, G58 and G10-U60 wobble base pair. Both the protein and the RNA exhibit a partially induced fit for binding, where loops in the protein and the internal loop in the RNA become more ordered upon complex formation. The specific interactions formed between loops on L30 and the internal loop on the mRNA constitute a novel loop-loop recognition motif where an intimate RNA-protein interface is formed between regions on both molecules that lack regular secondary structure.     

Interactions of cations with RNA loop-loop complexes

RNA loop-loop interactions are essential in many biological processes, including initiation of RNA folding into complex tertiary shapes, promotion of dimerization, and viral replication. In this article, we examine interactions of metal ions with five RNA loop-loop complexes of unique biological significance using explicit-solvent molecular-dynamics simulations. These simulations revealed the presence of solvent-accessible tunnels through the major groove of loop-loop interactions that attract and retain cations. Ion dynamics inside these loop-loop complexes were distinctly different from the dynamics of the counterion cloud surrounding RNA and depend on the number of basepairs between loops, purine sequence symmetry, and presence of unpaired nucleotides. The cationic uptake by kissing loops depends on the number of basepairs between loops. It is interesting that loop-loop complexes with similar functionality showed similarities in cation dynamics despite differences in sequence and loop size.     

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

Dicer is a member of the ribonuclease III enzyme family and processes double‐stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non‐canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double‐stranded RNA‐binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA‐binding surface. The second dsRNA binding domain at C‐terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem‐loop structure of the RNA substrate, suggesting the possibility that stem‐loop RNA‐guided gene silencing pathway exists in budding yeast.     

Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site

Loop-loop interactions among nucleic acids constitute an important form of molecular recognition in a variety of biological systems. In HIV-1, genomic dimerization involves an intermolecular RNA loop-loop interaction at the dimerization initiation site (DIS), a hairpin located in the 5' noncoding region that contains an autocomplementary sequence in the loop. Only two major DIS loop sequence variants are observed among natural viral isolates. To investigate sequence and structural constraints on genomic RNA dimerization as well as loop-loop interactions in general, we randomized several or all of the nucleotides in the DIS loop and selected in vitro for dimerization-competent sequences. Surprisingly, increasing interloop complementarity above a threshold of 6 bp did not enhance dimerization, although the combinations of nucleotides forming the theoretically most stable hexanucleotide duplexes were selected. Noncanonical interactions contributed significantly to the stability and/or specificity of the dimeric complexes as demonstrated by the overwhelming bias for noncanonical base pairs closing the loop and covariations between flanking and central loop nucleotides. Degeneration of the entire loop yielded a complex population of dimerization-competent sequences whose consensus sequence resembles that of wild-type HIV-1. We conclude from these findings that the DIS has evolved to satisfy simultaneous constraints for optimal dimerization affinity and the capacity for homodimerization. Furthermore, the most constrained features of the DIS identified by our experiments could be the basis for the rational design of DIS-targeted antiviral compounds.     

An insertion in loop L7 of human eosinophil-derived neurotoxin is crucial for its antiviral activity

The human eosinophil granule ribonuclease, eosinophil‐derived neurotoxin (EDN) has been shown to have antiviral activity against respiratory syncytial virus‐B (RSV‐B). Other closely related and more active RNases such as RNase A, onconase, and RNase k6 do not have any antiviral activity. A remarkable unique feature of EDN is a nine‐residue insertion in its carboxy‐terminal loop, L7 which is not present in RNase A, and differs in sequence from the corresponding loop in another eosinophil RNase, eosinophil cationic protein (ECP). ECP has a much lower antiviral activity as compared to EDN. The current study probed the role of loop L7 of EDN in its antiviral activity. Three residues in loop L7, Arg117, Pro120, and Gln122, which diverge between EDN, ECP, and RNase A, were mutated to alanine alone and in combination to generate single, double, and triple mutants. These mutants, despite having RNase activity had decreased antiviral activity towards RSV suggesting the involvement of loop L7 in the interaction of EDN with RSV. It appears that the mutations in loop L7 disrupt the interaction of protein with the viral capsid, thereby inhibiting its entry into the virions. The study demonstrates that besides the RNase activity, loop L7 is another important determinant for the antiviral activity of EDN. J. Cell. Biochem. 113: 3104–3112, 2012. © 2012 Wiley Periodicals, Inc.     

Mutational analysis of an RNA internal loop as a reactivity epitope for Escherichia coli ribonuclease III substrates

The enzymatic cleavage of double-stranded (ds) RNA is an obligatory step in the maturation and decay of many cellular and viral RNAs. The primary agents of dsRNA processing are members of the ribonuclease III (RNase III) superfamily, which are highly conserved in eukaryotic and bacterial cells. Escherichia coli RNase III participates in the maturation of the ribosomal RNAs and in the maturation and decay of cellular and phage mRNAs. E. coli RNase III-dependent cleavage events can regulate gene expression by controlling mRNA stability and translational activity. RNase III recognizes its substrates and selects the scissile phosphodiester(s) by recognizing specific RNA sequence and structural elements, termed reactivity epitopes. Some E. coli RNase III substrates contain an internal loop, in which is located the single scissile phosphodiester. The specific features of the internal loop that establish the pattern of single-strand cleavage are not known. A mutational analysis of the asymmetric [4 nt/5 nt] internal loop of the phage T7 R1.1 substrate reveals that cleavage reactivity is largely independent of internal loop sequence. Instead, the [4/5] asymmetry per se is the primary determinant of cleavage of a single bond within the 5 nt strand of the internal loop. The T7 R1.1 internal loop lacks elements of local tertiary structure, as revealed by sensitivity to cleavage by terbium ion and by the ability of the internal loop to destabilize a small model duplex. The internal loop functions as a discrete structural element in that the pattern of cleavage can be controlled by the specific type of asymmetry. The implications of these findings are discussed in light of RNase III substrate function as a gene regulatory element.     

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.     

Infectious Molecular Clones with the Nonhomologous Dimer Initiation Sequences Found in Different Subtypes of Human Immunodeficiency Virus Type 1 Can Recombine and Initiate a Spreading Infection In Vitro

Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.     

Kissing of the two predominant hairpin loops in the coxsackie B virus 3' untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis.

Higher-order RNA structures in the 3' untranslated region (3'UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3'UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V.I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3'UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3'UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3'UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.     

The UAA/GAN internal loop motif: a new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions

Analysis of aligned RNA sequences and high-resolution crystal structures has revealed a new RNA structural element, termed the UAA/GAN motif. Found in internal loops of the 23 S rRNA, as well as in RNase P RNA and group I and II introns, this six-nucleotide motif adopts a distinctive local structure that includes two base-pairs with non-canonical conformations and three conserved adenine bases, which form a cross-strand AAA stack in the minor groove. Most importantly, the motif invariably forms long-range tertiary contacts, as the AAA stack typically forms A-minor interactions and the flipped-out N nucleotide forms additional contacts that are specific to the structural context of each loop. The widespread presence of this motif and its propensity to form long-range contacts suggest that it plays a critical role in defining the architectures of structured RNAs.