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PubMed2006

Structure and function of RNA replication.

Contrary to their host cells, many viruses contain RNA as genetic material and hence encode an RNA-dependent RNA polymerase to replicate their genomes. This review discusses the present status of our knowledge on the structure of these enzymes and the mechanisms of RNA replication. The simplest viruses encode only the catalytic subunit of the replication complex, but other viruses also contribute a variable number of ancillary factors. These and other factors provided by the host cell play roles in the specificity and affinity of template recognition and the assembly of the replication complex. Usually, these host factors are involved in protein synthesis or RNA modification in the host cell, but they play roles in remodeling RNA-RNA, RNA-protein, and protein-protein interactions during virus RNA replication. Furthermore, viruses take advantage of and modify previous cell structural elements, frequently membrane vesicles, for the formation of RNA replication complexes.

Genome, Viral↗
PubMed2006

Long-distance RNA-RNA interactions in plant virus gene expression and replication.

The vast majority of plant and animal viruses have RNA genomes. Viral gene expression and replication are controlled by cis-acting elements in the viral genome, which have been viewed conventionally as localized structures. However, recent research has altered this perception and provided compelling evidence for cooperative activity involving distantly positioned RNA elements. This chapter focuses on viral RNA elements that interact across hundreds or thousands of intervening nucleotides to control translation, genomic RNA synthesis, and subgenomic mRNA transcription. We discuss evidence supporting the existence and function of the interactions, and speculate on the regulatory roles that such long-distance interactions play in the virus life cycle. We emphasize viruses in the Tombusviridae and Luteoviridae families in which long-distance interactions are best characterized, but similar phenomena in other viruses are also discussed. Many more examples likely remain undiscovered.

Gene Expression Regulation, Viral↗
PubMed2004

The conserved adenosine in helix 6 of Archaeoglobus fulgidus signal recognition particle RNA initiates SRP assembly.

The signal recognition particle (SRP) RNA helix 6 of archaea and eukaryotes is essential for the binding of protein SRP19 and the assembly of a functional complex. The conserved adenosine at the third position of the tetraloop of helix 6 (A149) is crucial for the binding of protein SRP19 in the mammalian SRP. Here we investigated the significance of the equivalent adenosine residue at position 159 (A159) of Archaeoglobus fulgidus SRP RNA. The A159 of A. fulgidus and A149 of human SRP RNA were changed to C, G or U, and fragments containing helix 6 or helices 6 and 8 were synthesized by run-off transcription with T7 RNA polymerase. The ability of recombinant A. fulgidus and human SRP19 to form ribonucleoprotein complexes was measured in vitro. The simultaneous presence of A149 and helix 8 is required for the high-affinity binding of SRP19 to the human SRP RNA. In contrast, A. fulgidus SRP19 binds to the SRP RNA fragments with high affinity irrespective of the nature of the nucleotide, demonstrating that A159 does not directly participate in protein binding. Instead, as indicated by the resistance of the wild-type A. fulgidus RNA towards digestion by RNase A, this residue allows the formation of a tightly folded RNA molecule. The high affinity between A.fulgidus SRP 19 and RNA molecules that contain both helices 6 and 8 suggests that A159 is likely to initiate archaeal SRP assembly by forming a conserved tertiary RNA-RNA interaction.

Adenosine↗
PubMed2006

Partition function and base pairing probabilities of RNA heterodimers.

BACKGROUND: RNA has been recognized as a key player in cellular regulation in recent years. In many cases, non-coding RNAs exert their function by binding to other nucleic acids, as in the case of microRNAs and snoRNAs. The specificity of these interactions derives from the stability of inter-molecular base pairing. The accurate computational treatment of RNA-RNA binding therefore lies at the heart of target prediction algorithms. METHODS: The standard dynamic programming algorithms for computing secondary structures of linear single-stranded RNA molecules are extended to the co-folding of two interacting RNAs. RESULTS: We present a program, RNAcofold, that computes the hybridization energy and base pairing pattern of a pair of interacting RNA molecules. In contrast to earlier approaches, complex internal structures in both RNAs are fully taken into account. RNAcofold supports the calculation of the minimum energy structure and of a complete set of suboptimal structures in an energy band above the ground state. Furthermore, it provides an extension of McCaskill's partition function algorithm to compute base pairing probabilities, realistic interaction energies, and equilibrium concentrations of duplex structures.

Journal Article↗
PubMed1995

Hybridization of the complementary mRNAs for P450c21 (steroid 21-hydroxylase) and tenascin-X is prevented by sequence-specific binding of nuclear proteins.

The CYP21 gene that encodes the steroid 21-hydroxylase, P450c21, is overlapped on the opposite strand of DNA by the TX-X gene encoding the extracellular matrix protein, tenascin-X. These transcripts contain perfectly complementary segments of 299 bases at their 3'-ends. As these genes are tandemly duplicated and are transcribed in the adrenal cortex, we investigated whether these self-complementary transcripts formed RNA-RNA hybrids in vivo. Formation of heterogeneous nuclear ribonucleoprotein complexes between nascent RNA transcripts and nuclear proteins might modulate such potential RNA-RNA interactions. Using a double RNase protection assay, we found that these RNAs form very small amounts of double-stranded RNA-RNA hybrids in adrenal cells in vivo. To understand why these mRNAs fail to hybridize in vivo, we studied the actions of nuclear proteins on the binding and annealing of their complementary regions in vitro. The nucleation of interstrand annealing was kinetically favored over binding and was efficiently promoted by nuclear extracts. However, RNA-RNA strand zippering was inhibited, suggesting that protein binding and/or stable RNA secondary structures contribute to discontiguous base pairing. Increasing concentrations of nuclear proteins increased the relative proportion of these RNAs in perfect RNase-resistant duplexes but reached only about 20% of the total available RNA strands at saturating concentrations of nuclear proteins. Preincubation of either of the two single-stranded RNAs with nuclear proteins strongly inhibited the nucleation step of annealing, whereas preincubation of both strands abolished the annealing. RNase footprinting of the wild type and mutagenized overlapping transcripts suggested that sequence-specific binding of nuclear proteins is limited to the 5'-half of each RNA strand. These results indicate that the transcription of complementary, opposite-strand RNAs is not a mechanism for the regulation of the abundance of adrenal P450c21 mRNA and suggest that nuclear proteins strongly interfere with interstrand RNA base pairing in vitro as well as in vivo.

Base Composition↗
PubMed2005

A yeast RNA-hybrid system for the detection of RNA-RNA interactions in vivo.

RNA-RNA interactions play a crucial role at many different levels of the cellular metabolism such as plasmid replication control, viral encapsidation, or transcriptional and translational regulation. Therefore, methods are necessary to investigate the molecular determinants of given interactions, including their stabilities, or to screen for new interacting partners. We designed an RNA-hybrid system in S. cerevisiae, based on the yeast three-hybrid system. In this setup, the activation of a reporter gene is dependent on the interaction of two RNAs. A loop-loop interaction similar to the dimerization initiation site of the HIV genome was used as a model system, demonstrating that in this novel RNA-hybrid system only cognate RNAs promote the activation of the reporter gene. Levels of reporter activation correlate well with interaction stabilities determined in vitro by UV melting analyses, suggesting that conditions used for the analysis of in vitro structural stabilities translate well into the intracellular environment. Furthermore, the system was applicable for a screen against a test library. Nine out of ten selected clones were identified as predicted interaction partners for the bait RNA. In summary, we present a yeast reporter system depending on RNA-RNA interactions, which can be used alternatively for analysis of known interactions or for screening libraries in search for new interaction partners.

Base Pairing↗
PubMed2003

A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits.

Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.

Base Sequence↗
PubMed2005

Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation.

Internal ribosome entry site (IRES) elements consist of highly structured RNA regions that determine internal initiation of translation. We have previously shown that the foot-and-mouth disease virus (FMDV) IRES contains a GNRA tetraloop spanning residues G178UAA181. Here we show that tertiary RNA interactions dependent on the GNRA motif determine the structural organization of the central domain. By using mutational analysis in combination with RNA probing, we have identified distant reciprocal interactions between the GNRA motif and the invariant region G240CACG244, termed motif A. Mutations in motif A caused a decrease in IRES activity as severe as the GUAG substitution in the GNRA motif. Substitutions in either GNRA or motif A sequences induced a common reorganization around the conserved R199AAA202 stem-loop, suggesting that the latter contributes to stabilize the GNRA-motif A interaction. This finding was also consistent with a significant increase in the efficiency of RNA-RNA interactions determined in gel shift assays using as probe the hairpin that contains the GNRA motif compared to a transcript encompassing the entire apical region of the central domain. Thus, we propose that the central domain of the FMDV IRES contains a structural conformation essential for IRES activity stabilized by a tertiary contact involving residues in the GNRA tetraloop and motif A conserved sequences.

Base Sequence↗
PubMed2003

Long-range RNA-RNA interaction between the 5' nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation.

Hepatitis C virus (HCV) is a positive-sense RNA virus approximately 9600 bases long. An internal ribosomal entry site (IRES) spans the 5' nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5'NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5'NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.

5' Untranslated Regions↗
PubMed2006

Frequency of RNA-RNA interaction in a model of the RNA World.

The RNA World model for prebiotic evolution posits the selection of catalytic/template RNAs from random populations. The mechanisms by which these random populations could be generated de novo are unclear. Non-enzymatic and RNA-catalyzed nucleic acid polymerizations are poorly processive, which means that the resulting short-chain RNA population could contain only limited diversity. Nonreciprocal recombination of smaller RNAs provides an alternative mechanism for the assembly of larger species with concomitantly greater structural diversity; however, the frequency of any specific recombination event in a random RNA population is limited by the low probability of an encounter between any two given molecules. This low probability could be overcome if the molecules capable of productive recombination were redundant, with many nonhomologous but functionally equivalent RNAs being present in a random population. Here we report fluctuation experiments to estimate the redundancy of the set of RNAs in a population of random sequences that are capable of non-Watson-Crick interaction with another RNA. Parallel SELEX experiments showed that at least one in 10(6) random 20-mers binds to the P5.1 stem-loop of Bacillus subtilis RNase P RNA with affinities equal to that of its naturally occurring partner. This high frequency predicts that a single RNA in an RNA World would encounter multiple interacting RNAs within its lifetime, supporting recombination as a plausible mechanism for prebiotic RNA evolution. The large number of equivalent species implies that the selection of any single interacting species in the RNA World would be a contingent event, i.e., one resulting from historical accident.

Bacillus subtilis↗
PubMed2006

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome.

Current models of the core of the spliceosome include a network of RNA-RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8-RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome's RNA core.

Base Sequence↗
PubMed2006

Conformational organization of the 3' untranslated region in the tomato bushy stunt virus genome.

The 3' untranslated regions (UTRs) of positive-strand RNA viruses often form complex structures that facilitate various viral processes. We have examined the RNA conformation of the 352 nucleotide (nt) long 3' UTR of the Tomato bushy stunt virus (TBSV) genome with the goal of defining both local and global structures that are important for virus viability. Gel mobility analyses of a 3'-terminal 81 nt segment of the 3' UTR revealed that it is able to form a compact RNA domain (or closed conformation) that is stabilized by a previously proposed tertiary interaction. RNA-RNA gel shift assays were used to provide the first physical evidence for the formation of this tertiary interaction and revealed that it represents the dominant or "default" structure in the TBSV genome. Further analysis showed that the tertiary interaction involves five base pairs, each of which contributes differently to overall complex stability. Just upstream from the 3'-terminal domain, a long-distance RNA-RNA interaction involving 3' UTR sequences was found to be required for efficient viral RNA accumulation in vivo and to also contribute to the formation of the 3'-terminal domain in vitro. Collectively, these results provide a comprehensive overview of the conformational and functional organization of the 3' UTR of the TBSV genome.

3' Untranslated Regions↗
PubMed2006

Long-distance RNA-RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis.

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Base Sequence↗
PubMed2005

Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein.

The interaction between box C/D small nucleolar (sno)RNAs and the 15.5K protein nucleates snoRNP assembly. Many eukaryotic snoRNAs contain two potential binding sites for this protein, only one of which appears to be utilized in vivo. The binding site conforms to the consensus for a kink-turn motif. We have investigated the molecular basis for selection of one potential site over the other using in vitro mobility shift assays and nucleotide analog interference mapping of Xenopus U25 snoRNA and of a circularly permuted form. We find that preferential binding of human 15.5K is not dependent on the proximity of RNA ends, but instead appears to require a structural context beyond the kink-turn itself. Direct analysis of the energetic contributions to binding made by 18 functional groups within the kink-turn identified both backbone atoms and base functionalities as key for interaction. An intramolecular RNA-RNA contact via a 2'-hydroxyl may supercede a putative Type I A-minor interaction in stabilizing the RNA-protein complex.

Animals↗
PubMed2006

tRNA regulation of gene expression: interactions of an mRNA 5'-UTR with a regulatory tRNA.

Many genes encoding aminoacyl-tRNA synthetases and other amino acid-related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5'-untranslated region (5'-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the "Specifier Loop" of the 5'-UTR. For the 5'-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules ("Common" and "Specifier") would reconstitute the Specifier Loop region of the 5'-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental Kds of 27.6 +/- 1.0 microM and 10.5 +/- 0.7 microM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5'-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNA(Gly) (ASL(Gly)(GCC)) specifically and with a significant affinity (Kd = 20.2 +/- 1.4 microM). Thus, the bimolecular 5'-UTR and ASL(Gly)(GCC) models mimic the RNA-RNA interaction required for T box gene regulation in vivo.

5' Untranslated Regions↗
PubMed2004

Analysis of a long-range interaction between conserved domains of human telomerase RNA.

Telomerase is a ribonucleoprotein complex responsible for maintaining telomere length of eukaryotic chromosomes. Human telomerase has two main components, the human telomerase reverse transcriptase and the human telomerase RNA (hTR). Two domains of hTR essential for telomerase activity are the template domain, comprised of an 11-nt templating and alignment sequence, and the CR4/CR5 domain. Highly conserved residues in the CR4/CR5 domain form the stem-loop P6.1, which is important for assembly and activity of mammalian telomerase. Here, we have determined that stem-loop P6.1 can participate in a long-range RNA-RNA interaction with the template region of hTR. We characterized this interaction through mobility shift assays, mutation analysis, and UV cross-linking experiments. Mutation analysis revealed that the P6.1 loop nucleotides participate in the interaction with the template. The site of interaction at the template domain was determined via UV cross-linking experiments. These data show that an RNA-RNA interaction exists between two highly conserved regions of hTR that are critical for the higher order folding of telomerase RNA. This interaction argues for the proximity of the template and the CR4/CR5 domain, and provides the basis for a revised model of hTR, partitioning the RNA into a catalytic domain and a localization domain.

Base Pairing↗
PubMed2003

Visualization of membrane RNAs.

Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of approximately 2-8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA-RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world.

Binding Sites↗
PubMed2006

Analysis of a 3'-translation enhancer in a tombusvirus: a dynamic model for RNA-RNA interactions of mRNA termini.

Tomato bushy stunt virus is a (+)-strand RNA virus that is neither 5'-capped nor 3'-polyadenylated. Translation of viral proteins is instead mediated by an RNA element, the 3'-cap-independent translational enhancer (3'CITE), which is located in its 3' untranslated region (UTR). The 3'CITE is proposed to recruit the translational machinery to the viral message, while a long-distance RNA-RNA interaction between the 3'CITE and 5' UTR is thought to deliver the 43S ribosomal subunit to the 5' end of the viral mRNA. Here we provide the first evidence that the 5' UTR and 3'CITE interact physically. Mutational analysis showed that formation of this RNA-RNA interaction in vitro correlates well with efficient translation in vivo, thus supporting its functional relevance. Other analyses of the 3'CITE confirmed an overall Y-shaped RNA secondary structure and demonstrated the importance of numerous minor structural features for efficient translation of viral mRNAs. Functional studies on the role of the 5' UTR revealed that despite the absence of a cap structure, 43S subunits load at the very 5' end and scan in a 3' direction. These results indicate that the 5'-3' RNA-RNA interaction is likely disrupted by scanning ribosomal subunits and suggest a dynamic model for the interaction of mRNA termini during active translation.

3' Untranslated Regions↗