Long-distance Base Pairing Research Articles

A Stem-Loop Structure in Potato Leafroll Virus Open Reading Frame 5 (ORF5) Is Essential for Readthrough Translation of the Coat Protein ORF Stop Codon 700 Bases Upstream.

Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon. Mutations predicted to disrupt and restore the base of the distal stem-loop structure prevented and restored stop codon readthrough. Motifs in the downstream readthrough element (DRTE) are predicted to base pair to a site within 27 nucleotides (nt) of the CP ORF stop codon. Consistent with a requirement for this base pairing, the DRTE of Cereal yellow dwarf virus was not compatible with the stop codon-proximal element of PLRV in facilitating readthrough. Moreover, deletion of the complementary tract of bases from the stop codon-proximal region or the DRTE of PLRV prevented readthrough. In contrast, the distance and sequence composition between the two domains was flexible. Mutants deficient in RTP translation moved long distances in plants, but fewer infection foci developed in systemically infected leaves. Selective 2'-hydroxyl acylation and primer extension (SHAPE) probing to determine the secondary structure of the mutant DRTEs revealed that the functional mutants were more likely to have bases accessible for long-distance base pairing than the nonfunctional mutants. This study reveals a heretofore unknown combination of RNA structure and sequence that reduces stop codon efficiency, allowing translation of a key viral protein.IMPORTANCE Programmed stop codon readthrough is used by many animal and plant viruses to produce key viral proteins. Moreover, such "leaky" stop codons are used in host mRNAs or can arise from mutations that cause genetic disease. Thus, it is important to understand the mechanism(s) of stop codon readthrough. Here, we shed light on the mechanism of readthrough of the stop codon of the coat protein ORFs of viruses in the Luteoviridae by identifying the amino acids inserted at the stop codon and RNA structures that facilitate this "leakiness" of the stop codon. Members of the Luteoviridae encode a C-terminal extension to the capsid protein known as the readthrough protein (RTP). We characterized two RNA domains in Potato leafroll virus (PLRV), located 600 to 700 nucleotides apart, that are essential for efficient RTP translation. We further determined that the PLRV readthrough process involves both local structures and long-range RNA-RNA interactions. Genetic manipulation of the RNA structure altered the ability of PLRV to translate RTP and systemically infect the plant. This demonstrates that plant virus RNA contains multiple layers of information beyond the primary sequence and extends our understanding of stop codon readthrough. Strategic targets that can be exploited to disrupt the virus life cycle and reduce its ability to move within and between plant hosts were revealed.

The Cellular Decapping Activators LSm1, Pat1, and Dhh1 Control the Ratio of Subgenomic to Genomic Flock House Virus RNAs

Positive-strand RNA viruses depend on recruited host factors to control critical replication steps. Previously, it was shown that replication of evolutionarily diverse positive-strand RNA viruses, such as hepatitis C virus and brome mosaic virus, depends on host decapping activators LSm1-7, Pat1, and Dhh1 (J. Diez et al., Proc. Natl. Acad. Sci. U. S. A. 97:3913-3918, 2000; A. Mas et al., J. Virol. 80:246 -251, 2006; N. Scheller et al., Proc. Natl. Acad. Sci. U. S. A. 106:13517-13522, 2009). By using a system that allows the replication of the insect Flock House virus (FHV) in yeast, here we show that LSm1-7, Pat1, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.

Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5′ splice site, which greatly increases use of this 5′ splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5′- and 3′-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3′-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5′ ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5′ end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3′ cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Role of the 5′ TAR Stem−Loop and the U5-AUG Duplex in Dimerization of HIV-1 Genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).

The 3' cap-independent translation element of Barley yellow dwarf virus binds eIF4F via the eIF4G subunit to initiate translation.

The 3' cap-independent translation element (BTE) of Barley yellow dwarf virus RNA confers efficient translation initiation at the 5' end via long-distance base pairing with the 5'-untranslated region (UTR). Here we provide evidence that the BTE functions by recruiting translation initiation factor eIF4F. We show that the BTE interacts specifically with the cap-binding initiation factor complexes eIF4F and eIFiso4F in a wheat germ extract (wge). In wge depleted of cap-interacting factors, addition of eIF4F (and to a lesser extent, eIFiso4F) allowed efficient translation of an uncapped reporter construct (BLucB) containing the BTE in its 3' UTR. Translation of BLucB required much lower levels of eIF4F or eIFiso4F than did a capped, nonviral mRNA. Both full-length eIF4G and the carboxy-terminal half of eIF4G lacking the eIF4E binding site stimulated translation to 70% of the level obtained with eIF4F, indicating a minor role for the cap-binding protein, eIF4E. In wge inhibited by either BTE in trans or cap analog, eIF4G alone restored translation nearly as much as eIF4F, while addition of eIF4E alone had no effect. The BTE bound eIF4G (Kd = 177 nm) and eIF4F (Kd = 37 nm) with high affinity, but very weakly to eIF4E. These interactions correlate with the ability of the factors to facilitate BTE-mediated translation. These results and previous observations are consistent with a model in which eIF4F is delivered to the 5' UTR by the BTE, and they show that eIF4G, but not eIF4E, plays a major role in this novel mechanism of cap-independent translation.

Translation mechanisms involving long-distance base pairing interactions between the 5′ and 3′ non-translated regions and internal ribosomal entry are conserved for both genomic RNAs of Blackcurrant reversion nepovirus

One of the mechanisms of functioning for viral cap-independent translational enhancers (CITEs), located in 3′ non-translated regions (NTRs), is 3′ NTR–5′ leader long-distance base pairing. Previously, we have demonstrated that the RNA2 3′ NTR of Blackcurrant reversion nepovirus (BRV) contains a CITE, which must base pair with the 5′ NTR to facilitate translation. Here we compared translation strategies employed by BRV RNA1 and RNA2, by using mutagenesis of the BRV NTRs in firefly luciferase reporter mRNA, in plant protoplasts. Translation mechanisms, based on 3′ CITEs, 5′ NTR–3′ NTR base pairing and poly(A) tail-stimulation, were found conserved between RNA1 and RNA2. The 40S ribosomal subunit entry at the RNA1 leader occurred, at least partly, via an internal ribosomal entry site (IRES). Two RNA1 leader segments complementary to plant 18S rRNA enhanced translation. A model for BRV RNAs translation, involving IRES-dependent 40S subunit recruitment and long-distance 5′ NTR–3′ NTR base pairing, is discussed.

Oscillating kissing stem–loop interactions mediate 5′ scanning-dependent translation by a viral 3′-cap-independent translation element

The 3'-untranslated regions (UTRs) of a group of novel uncapped viral RNAs allow efficient translation initiation at the 5'-proximal AUG. A well-characterized model is the Barley yellow dwarf virus class of cap-independent translation elements (BTE). It facilitates translation by forming kissing stem-loops between the BTE in the 3'-UTR and a BTE-complementary loop in the 5'-UTR. Here we investigate the mechanisms of the long-distance interaction and ribosome entry on the RNA. Upstream AUGs or 5'-extensions of the 5'-UTR inhibit translation, indicating that, unlike internal ribosome entry sites in many viral RNAs, the BTE relies on 5'-end-dependent ribosome scanning. Cap-independent translation occurs when the kissing sites are moved to different regions in either UTR, including outside of the BTE. The BTE can even confer cap-independent translation when fused to the 3'-UTR of a reporter RNA harboring dengue virus sequences that cause base-pairing between the 3'- and 5'-ends. Thus, the BTE serves as a functional sensor to detect sequences capable of long-distance base-pairing. We propose that the kissing interaction is repeatedly disrupted by the scanning ribosome and re-formed in an oscillating process that regulates ribosome entry on the RNA.

Structural Constraints and Mutational Bias in the Evolutionary Restoration of a Severe Deletion in RNA Phage MS2

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations.

The 3' untranslated region of tobacco necrosis virus RNA contains a barley yellow dwarf virus-like cap-independent translation element.

RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.

A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA.

Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.

Regulated HIV-2 RNA dimerization by means of alternative RNA conformations.

The dimer initiation site (DIS) hairpin of the HIV-2 untranslated leader RNA mediates in vitro dimerization through 'loop-loop kissing' of a loop-exposed palindrome sequence. Premature RNA dimerization must be prevented during the retroviral life cycle. A regulatory mechanism has been proposed for the HIV-1 leader RNA that can adopt an alternative conformation in which the DIS motif is effectively masked by long-distance base pairing with upstream leader sequences. We now report that HIV-2 RNA dimerization is also regulated. Sequestering of the DIS motif by base pairing interactions with downstream leader sequences mediates a switch to a dimerization-impaired conformation. The existence of two alternative conformations of the HIV-2 leader RNA is supported by UV melting experiments. Furthermore, the equilibrium between the two conformations can be shifted by annealing of antisense oligonucleotides or by deletion of certain leader regions. These measures have a profound impact on the dimerization properties of the transcript, demonstrating a mutual exclusivity between the alternative conformation and dimerization, similar to what has been described for the HIV-1 leader. The overall resemblance in regulation of HIV-1 and HIV-2 RNA dimerization suggests that a similar mechanism may be operating in other lentiviruses and perhaps all retroviridae.

Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA.

The HIV-1 untranslated leader RNA can adopt two mutually exclusive conformations that represent alternative secondary structures. This leader RNA can fold either an extended duplex through long-distance base pairing or a branched conformation in which the RNA locally folds into hairpin structures. Both leader RNA conformations have the TAR hairpin in common, which forms the extreme 5' end of all HIV-1 transcripts. We report that truncation of the TAR hairpin shifts the equilibrium between the two RNA conformations away from the thermodynamically favored long-distance interaction. However, the equilibrium is partially restored in response to the cations Na(+) and Mg(2+). The transcripts with mutant TAR structures allowed us to investigate conditions affecting the competition between the alternative conformations of the HIV-1 leader RNA. We also demonstrate that the change in conformation of the leader RNA due to TAR truncations severely affects formation of the HIV-1 RNA dimer.

Base-Pairing between Untranslated Regions Facilitates Translation of Uncapped, Nonpolyadenylated Viral RNA

Translationally competent mRNAs form a closed loop via interaction of initiation factors with the 5′ cap and poly(A) tail. However, many viral mRNAs lack a cap and/or a poly(A) tail. We show that an uncapped, nonpolyadenylated plant viral mRNA forms a closed loop by direct base-pairing (kissing) of a stem loop in the 3′ untranslated region (UTR) with a stem loop in the 5′ UTR. This allows a sequence in the 3′ UTR to confer translation initiation at the 5′-proximal AUG. This base-pairing is also required for replication. Unlike other cap-independent translation mechanisms, the ribosome enters at the 5′ end of the mRNA. This remarkably long-distance base-pairing reveals a novel mechanism of cap-independent translation and means by which mRNA UTRs can communicate.

Translational control by delayed RNA folding: identification of the kinetic trap.

The maturation or A-protein gene of single-stranded RNA phage MS2 is preceded by a 130-nt long untranslated leader. When MS2 RNA folding is at equilibrium, the gene is untranslatable because the leader adopts a well-defined cloverleaf structure in which the Shine-Dalgarno (SD) sequence of the maturation gene is taken up in long-distance base pairing with an upstream complementary sequence (UCS). Synthesis of the A-protein takes place transiently while the RNA is synthesized from the minus strand. This requires that formation of the inhibitory cloverleaf is slow. In vitro, the folding delay was on the order of minutes. Here, we present evidence that this postponed folding is caused by the formation of a metastable intermediate. This intermediate is a small local hairpin that contains the UCS in its loop, thereby preventing or slowing down its pairing with the SD sequence. Mutants in which the small hairpin could not be formed made no detectable amounts of A-protein and were barely viable. Apparently, here the cloverleaf formed quicker than ribosomes could bind. On the other hand, mutants in which the small intermediary hairpin was stabilized produced more A-protein than wild type and were viable. One hardly growing mutant that could not form the metastable hairpin and did not make detectable amounts of A-protein was evolved. The emerging pseudo-revertant had selected two second site repressor mutations that allowed reconstruction of a variant of the metastable intermediate. The pseudo-revertant had also regained the capacity to produce the A-protein.

Two alternating structures of the HIV-1 leader RNA.

In this study we demonstrate that the HIV-1 leader RNA exists in two alternative conformations, a branched structure consisting of several well-known hairpin motifs and a more stable structure that is formed by extensive long-distance base pairing. The latter conformation was first identified as a compactly folded RNA that migrates unusually fast in nondenaturing gels. The minimally required domains for formation of this conformer were determined by mutational analysis. The poly(A) and DIS regions of the leader are the major determinants of this RNA conformation. Further biochemical characterization of this conformer revealed that both hairpins are disrupted to allow extensive long-distance base pairing. As the DIS hairpin is known to be instrumental for formation of the HIV-1 RNA dimer, the interplay between formation of the conformer and dimerization was addressed. Formation of the conformer and the RNA dimer are mutually exclusive. Consequently, the conformer must rearrange into a branched structure that exposes the dimer initiation signal (DIS) hairpin, thus triggering formation of the RNA dimer. This structural rearrangement is facilitated by the viral nucleocapsid protein NC. We propose that this structural polymorphism of the HIV-1 leader RNA acts as a molecular switch in the viral replication cycle.