Search

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure., (© 2024 Chen et al.)

Oncogenesis involves continuous genetic alterations that lead to compromised cellular integrity and immortal cell fate. The cells remain under excessive stress due to endo- and exogenous influences. Human Satellite III long noncoding RNA (SatIII lncRNA) is a key regulator of the global cellular stress response, although its function is poorly explained in cancers. The principal regulator of cancer meshwork is tumor protein p53, which if altered may result in chemoresistance. The heat shock factor 1 (HSF1) being a common molecule between the oncogenic control and global cellular stress acts as an oncogene as well as transcribes SatIII upon heat shock. This prompted us to determine the structure of SatIII RNA and establish the association between SatIII-HSF1-p53. We determined the most stable structure of SatIII RNA with the least energy of - 115.7 kcal/mol. Also, we observed a possible interaction of p53 with SatIII and HSF1 using support vector machine (SVM) algorithm for predicting RNA-protein interaction (RPI). Further, we employ the STRING database to understand if p53 is an interacting component of the nuclear stress bodies (nSBs). A precise inference was drawn from molecular docking which confirmed the interaction of SatIII-HSF1-p53, where a mutated p53 resulted in an altered DNA-binding property with the SatIII molecule. This study being first of its kind infers p53 to be a possible integral component of the nSBs, which may regulate cellular stress response during cancer progression in the presence of HSF1 and SatIII. An extended research on the regulations of SatIII and p53 may open new avenues in the field of apoptosis in cancer and the early approach of molecular targeting., (© 2019 Wiley Periodicals, Inc.)

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3'-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The 356 nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5' sequences from a second TCV satRNA (satD) and 3' sequences derived from TCV. SHAPE structure mapping revealed that 76 nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38-39 nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38 nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38-39 nt hairpin, sufficient for overall structural stability of the 5' end of satC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

RNA FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA FISH protocol that we developed in order to study the expression and localization of satellite III RNAs. This specific class of non-coding RNAs is expressed in response to various cellular stresses including heat shock. This protocol is based on the use of a biotinylated LNA probe subsequently detected by a streptavidin-Alexa Fluor(®) 488 conjugate. A protocol allowing efficient coupling of RNA FISH and protein detection by immunofluorescence is also described in this chapter.

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.

Satellite RNAs associated with Bamboo mosaic virus (satBaMVs) depend on BaMV for replication and encapsidation. Certain satBaMVs isolated from natural fields significantly interfere with BaMV replication. The 5' apical hairpin stem loop (AHSL) of satBaMV is the major determinant in interference with BaMV replication. In this study, by in vivo competition assay, we revealed that the sequence and structure of AHSL, along with specific nucleotides (C(60) and C(83)) required for interference with BaMV replication, are also involved in replication competition among satBaMV variants. Moreover, all of the 5' ends of natural BaMV isolates contain the similar AHSLs having conserved nucleotides (C(64) and C(86)) with those of interfering satBaMVs, suggesting their co-evolution. Mutational analyses revealed that C(86) was essential for BaMV replication, and that replacement of C(64) with U reduced replication efficiency. The non-interfering satBaMV interfered with BaMV replication with the BaMV-C64U mutant as helper. These findings suggest that two cytosines at the equivalent positions in the AHSLs of BaMV and satBaMV play a crucial role in replication competence. The downregulation level, which is dependent upon the molar ratio of interfering satBaMV to BaMV, implies that there is competition for limited replication machinery.

Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta.

Viroids and satellite RNAs, which are the smallest infectious agents in plants, have noncoding RNA genomes and characteristic secondary structures. Some satellite RNAs (satRNAs) cause disease symptoms that are different from those induced by their helper virus. This phenomenon has been implicated in RNA silencing of host gene(s) as a result of sequence identity or complementarity between satRNAs and host RNAs. To investigate the effects of satRNA sequence on direct coincident interference with host gene expression, we developed a transient RNA silencing assay using protoplasts. With this protoplast system, we can induce various forms and lengths of silencing inducers at various concentrations to uniform cells without viral infection, and then we can use the satRNA-treated protoplasts in further analyses such as real-time RT-PCR and northern blot hybridization analyses to investigate whether the satRNA-induced symptoms are due to down-regulation of the target gene expression.

Lucerne transient streak virus (LTSV, genus Sobemovirus) supports the replication and encapsidation of a 322 nt untranslated small-circular RNA (scLTSV). Since scLTSV does not code for any proteins or share sequence similarity with its helper virus (LTSV), it is presumed that it uses structural and sequence motifs to signal the helper virus (and host) machinery for its replication and encapsidation. Insertion and deletion mutations were introduced at various locations within the scLTSV molecule. Our results showed that most mutants were not infectious, with only two exceptions, a (-1) nucleotide deletion and a 9 nt, palindromic insertion mutant which preserved the overall rod-like structure of the scLTSV. Sequence analysis of cDNA clones revealed that the palindromic sequence was replicated for up to 12 days of infection, before the sequence reverted back to its wild-type form. Our results indicate that scLTSV has an optimal sequence and secondary structure for replication, movement and/or packaging within the LTSV helper virus.

The biological and molecular properties of a novel satellite RNA (satRNA L) associated with tomato bushy stunt virus (TBSV) are described. satRNA L consisted of a linear single-stranded RNA of 615 nt, lacked significant open reading frames (ORFs) and had no sequence identity with the helper genome other than in the 5'-proximal 7 nt and in a central region that is also conserved in all tombusvirus genomic, defective interfering and satellite RNAs. Secondary-structure analysis showed the presence of high-order domains similar to those described for other tombusvirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV under any of the temperature conditions tested. A full-length cDNA clone was constructed and used in co-inoculations with transcripts of carnation Italian ringspot virus (CIRV) and cymbidium ringspot virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV-CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region was in the 5'-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region was in the ORF1-encoded protein where a more efficient satRNA L-binding domain is suggested to be present in CIRV.

Satellite RNAs (satRNAs) and satellite viruses depend on the replicase complexes provided by their cognate helper viruses and host plants for replication, pretending that they are part of the viral genomes. Although satRNAs and satellite viruses do not share significant nucleotide sequence similarity with the helper viruses, the essential cis-acting elements recognized by the replicase complexes must reside on their genomes, acting as the mimicry for the molecular pretenders. By understanding how this molecular mimicry deceives the helper viruses into supporting the satellites, a significant amount of knowledge of the basic requirements and mechanisms for replication of viruses and satellites has been obtained. Here we review the recent advances in understanding the effects of the cis elements at the termini of satRNAs and satellite viruses on their accumulation. Several well-characterized satellite/helper virus systems, representing the non-coding short satRNAs, mRNA-type long satRNAs, circular satRNAs and satellite viruses, are compared and contrasted. It is concluded that different satellites may adopt different strategies to exploit the replication/transcription/translation machineries of their helper viruses, and different mimicries may be implemented by the same molecular pretender for different biological functions.

The 3'-untranslated region (UTR) of RNA genomes of viruses and satellite RNAs plays essential roles in viral replication and transcription. The structural features of the 3'-UTR of the satellite RNA of Bamboo mosaic virus (satBaMV) involved in its replication were analyzed in this study. By the use of enzymatic probing, the secondary structure of satBaMV 3'-UTR was confirmed to comprise two small stem-loops (SLA and SLB), one large stem-loop (SLC), and a poly(A) tail of mainly 75-200 adenylate residues, which is similar to those on the genomic RNA of the helper virus, BaMV. Five sets of mutants of satBaMV were constructed to analyze the biological functions of the structural elements of the 3'-UTR. The data revealed that both the polyadenylation signal and poly(A) tail are required for satBaMV RNA replication. The structural conservation of SLA, SLB, and SLC is also important for efficient satBaMV accumulation, whereas the nucleotides in these regions may also possess sequence-specific functions. In contrast to the requirement for the accumulation of BaMV genomic RNA, mutations in the conserved hexanucleotide (ACCUAA) in the loop region of SLC had limited effect on the accumulation of satBaMV RNA. In addition, replacing the 5'-, 3'-UTR, or both regions of satBaMV by those of BaMV greatly decreased the accumulation of satBaMV RNA. Taken together, these data indicate that satBaMV might have adopted a 3'-UTR structure similar to that of BaMV but may have evolved distinct features for its efficient replication.

Carrot motley dwarf (CMD) is known to result from a mixed infection by two viruses, the polerovirus Carrot red leaf virus and one of the umbraviruses Carrot mottle mimic virus or Carrot mottle virus. Some umbraviruses have been shown to be associated with small satellite (sat) RNAs, but none have been reported for the latter two. A CMD-affected parsley plant was used for sap transmission to test plants, that were used for dsRNA isolation. The presence of a 0.8-kbp dsRNA indicated the occurrence of a hitherto unrecognized satRNA associated with CMD. The satRNAs of the CMD isolate from parsley and an isolate from carrot have been sequenced and showed 94% sequence identity. Nucleotide sequences and putative translation products had no significant similarities to GenBank entries. To our knowledge, this is the first report of satRNAs associated with CMD.

The genomes of positive-strand RNA viruses undergo conformational shifts that complicate efforts to equate structures with function. We have initiated a detailed analysis of secondary and tertiary elements within the 3' end of Turnip crinkle virus (TCV) that are required for viral accumulation in vivo. MPGAfold, a massively parallel genetic algorithm, suggested the presence of five hairpins (H4a, H4b, and previously identified hairpins H4, H5, and Pr) and one H-type pseudoknot (Psi(3)) within the 3'-terminal 194 nucleotides (nt). In vivo compensatory mutagenesis analyses confirmed the existence of H4a, H4b, Psi(3) and a second pseudoknot (Psi(2)) previously identified in a TCV satellite RNA. In-line structure probing of the 194-nt fragment supported the coexistence of H4, H4a, H4b, Psi(3) and a pseudoknot that connects H5 and the 3' end (Psi(1)). Stepwise replacements of TCV elements with the comparable elements from Cardamine chlorotic fleck virus indicated that the complete 142-nt 3' end, and subsets containing Psi(3), H4a, and H4b or Psi(3), H4a, H4b, H5, and Psi(2), form functional domains for virus accumulation in vivo. A new 3-D molecular modeling protocol (RNA2D3D) predicted that H4a, H4b, H5, Psi(3), and Psi(2) are capable of simultaneous existence and bears some resemblance to a tRNA. The related Japanese iris necrotic ring virus does not have comparable domains. These results provide a framework for determining how interconnected elements participate in processes that require 3' untranslated region sequences such as translation and replication.

The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.

Satellite RNAs associated with Bamboo mosaic virus (satBaMV) exhibit different phenotypes. Some isolates could reduce the accumulation of BaMV RNA and attenuate the BaMV-induced symptoms in co-inoculated plants. The determinants of the downregulation of BaMV replication were mapped in the 5' hypervariable region of satBaMV, which folds into a conserved apical hairpin stem loop (AHSL) structure comprising an apical loop and two internal loops, as evidenced by enzymatic probing. We also demonstrated that the integrity of the AHSL structure of interfering satBaMV was essential for the interference of BaMV accumulation. Concurrent analyses of natural satBaMV isolates revealed that all of the interfering isolates contained the same structures and sequences in the internal loops. Further, refined analyses indicated that, besides the AHSL structure, specific nucleotides in the internal loops play a crucial role in the downregulation, which implies that they may be required for the interaction of viral/cellular factors in this process.

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

RNA can adopt different conformations in response to changes in the metabolic status of cells, which can regulate processes such as transcription, translation, and RNA cleavage. We previously proposed that an RNA conformational switch in an untranslated satellite RNA (satC) of Turnip crinkle virus (TCV) regulates initiation of minus-strand synthesis (G. Zhang, J. Zhang, A. T. George, T. Baumstark, and A. E. Simon, RNA 12:147-162, 2006). This model was based on the lack of phylogenetically inferred hairpins or a known pseudoknot in the "preactive" structure assumed by satC transcripts in vitro. We now provide evidence that a second pseudoknot (Psi(2)), whose disruption reduces satC accumulation in vivo and enhances transcription by the TCV RNA-dependent RNA polymerase in vitro, stabilizes the preactive satC structure. Alteration of either Psi(2) partner caused nearly identical structural changes, including single-stranded-specific cleavages in the pseudoknot sequences and strong cleavages in a distal element previously proposed to mediate the conformational switch. These results indicate that the preactive structure identified in vitro has biological relevance in vivo and support a requirement for this alternative structure and a conformational switch in high-level accumulation of satC in vivo.

Background: Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs., Description: The Subviral RNA database is a web-based environment that facilitates the research and analysis of viroids, satellite RNAs, satellites viruses, the human hepatitis delta virus, and related RNA sequences. It integrates a large number of Subviral RNA sequences, their respective RNA motifs, analysis tools, related publication links and additional pertinent information (ex. links, conferences, announcements), allowing users to efficiently retrieve and analyze relevant information about these small RNA agents., Conclusion: With its design, the Subviral RNA Database could be considered as a fundamental building block for the study of these related RNAs. It is freely available via a web browser at the URL: http://subviral.med.uottawa.ca.

The full-length sequence of a satellite RNA (sat-RNA) of Beet black scorch virus isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to Chenopodium amaranticolor with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5- or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5' or the 3' termini. Therefore, sequences at both the 5' and the 3' ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5' end sequences and/or by internal initiation at the 3' ends of multimeric junctions.

Satellite (sat) RNAs are parasitic sub-viral RNA replicons found associated with certain positive-strand RNA viruses. Typical sat RNAs, such as those associated with members of the genus Tombusvirus, share little or no sequence identity with their helper virus genomes. Here, we have investigated a tombusvirus sat RNA and determined that it contains two functionally-relevant higher-order RNA domains, a T-shaped domain and a downstream domain, that are similar to elements shown previously to be present in the 5' untranslated regions (UTRs) of tombusvirus genomes. Although the two sat RNA domains showed only limited sequence identity with their viral counterparts, they were able to adopt comparably-folded RNA secondary structures. Interestingly, the relative spacing between the domains in the viral and satellite contexts was notably different. In the viral 5' UTR, the two domains are adjacent and separated by a small hairpin, however, in the sat RNA they are separated by a 137-nt long segment. Despite this distal modular arrangement, the two domains were found to be united spatially in the sat RNA through the formation of an RNA-RNA bridge. This co-localization facilitated an important inter-domain interaction and was essential for efficient helper-mediated sat RNA accumulation in protoplasts. These results indicate that the tombusvirus sat RNA and helper genome contain structurally and functionally equivalent RNA domains. It is proposed that the limited sequence identity observed between these corresponding higher-order RNA structures is related to a strategy that reduces the induction of gene silencing, which presumably would be detrimental to both viral and sat RNA replicons.

Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV.

Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.

Satellite RNA was sought in 51 isolates of Rice yellow mottle virus (RYMV) representative of the geographical, molecular and pathogenic variability of the virus in Africa. Three-quarters of the isolates from cultivated rice and wild gramineaceous hosts supported a satellite RNA. The prevalence of RYMV isolates that were associated with a satellite differed among regions, being c. 100% in West and Central Africa and c. 36% in East Africa. The RYMV satellite showed a low diversity as only seven of the 220 sequenced positions were variable. One insertion also occurred after serial host passages of the satellite. Two forms of the satellite differed by six substitutions forming three base pairs in one branch of the predicted RNA secondary structure. There was no evidence of intermediates between these two forms, but double-infection occurred. Each form had a specific geographical distribution: one occurred in Central Africa, the other elsewhere in Africa. There was no relation between the occurrence or the forms of the satellite and the phylogeny of the helper virus. The satellite was not involved in symptom modulation or ability to break host-plant resistances to the disease.

satC, a satellite RNA associated with Turnip crinkle virus (TCV), enhances the ability of the virus to colonize plants by interfering with stable virion accumulation (F. Zhang and A. E. Simon, unpublished data). Previous results suggested that the motif1-hairpin (M1H), a replication enhancer on minus strands, forms a plus-strand hairpin flanked by CA-rich sequence that may be involved in enhancing systemic infection (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003). In this study, sequence and structural requirements of the M1H were further assayed by replacing the 28-base M1H with 10 random bases and then subjecting the pool of satellite RNA to functional selection in plants. Unlike previous results with 28-base replacement sequences (G. Zhang and A. E. Simon, J. Mol. Biol. 326:35-48, 2003), only a few of the 10-base SELEX (systematic evolution of ligands by exponential enrichment) assay winners contained short motifs in their minus-sense orientation that were similar to TCV replication elements. However, all second- and third-round winning replacement sequences folded into hairpins flanked by CA-rich sequence predicted to be more stable on plus strands than minus strands. Plus strands of several of the most fit satellite RNAs contained insertions of CA-rich sequence at the base of their hairpins whose presence correlated with enhanced replication and reduced detection of virions. Deletion of the M1H resulted in no detectable virions despite very low satellite accumulation. These results support the hypothesis that a sequence-nonspecific plus-strand hairpin brings together flanking CA-rich sequences in the M1H region that confers fitness to satC by reducing the accumulation of stable virions.

The Neurospora Varkud Satellite (VS) RNA is capable of promoting a reversible self-cleavage reaction important for its replication pathway. In vivo the VS RNA performs a cis-cleavage reaction to generate monomeric length transcripts that are subsequently ligated to produce circular VS RNA. The predominant form of VS RNA observed in vivo is the closed circular form, though minimal VS ribozyme self-cleavage constructs lack detectable ligation activity. MFOLD analysis of the entire VS RNA sequence revealed an extended region 5' and 3' of the minimal self-cleaving region that could anneal to form a complementary helix, which we have termed helix 7. In full-length VS RNA, this helix appears to span over 40 bp of sequence and brings the 5'- and 3'-ends of the RNA into proximity for the ligation reaction. Here we report a variant of the VS ribozyme with an extended 5'- and 3'-terminus capable of forming a truncated helix 7 that promotes the ligation reaction in vitro. Through mutation and selection of this RNA we have identified a ribozyme containing two point mutations in the truncated helix 7 that ligates with >70% efficiency. These results show that an additional helical element absent in current VS ribozyme constructs is likely to be important for the ligation activity of VS RNA.

RNA replication enhancers are cis-acting elements that can stimulate replication or transcription of RNA viruses. Turnip crinkle virus (TCV) and satC, a parasitic RNA associated with TCV infections, contain stem-loop structures that are RNA replication enhancers (P. Nagy, J. Pogany, and A. E. Simon, EMBO J. 1999, 18, 5653-5665). We have found that replacement of 28 nt of the satC enhancer, termed the motif1-hairpin, with 28 randomized bases reduced satC accumulation 8- to 13-fold in Arabidopsis thaliana protoplasts. Deletion of single-stranded flanking sequences at either side of the hairpin also affected RNA accumulation with combined alterations at both sides of the hairpin showing the most detrimental effect in protoplasts. In vitro analysis with a partially purified TCV RdRp preparation demonstrated that the motif1-hairpin in its minus-sense orientation was able to stimulate RNA synthesis from the satC hairpin promoter (located at the 3' end of plus strands) by almost twofold. This level of RNA synthesis stimulation is approximately fivefold lower than that observed with a linear promoter, suggesting that a highly stable hairpin promoter is less responsive to the presence of the motif1-hairpin enhancer than a linear promoter. The motif1-hairpin in its plus-sense orientation was only 60% as active in enhancing transcription from the hairpin promoter. Since the motif1-hairpin is a hotspot for RNA recombination during plus-strand synthesis and since satC promoters located on the minus-strand are all short linear sequences, these findings support the hypothesis that the motif1-hairpin is primarily involved in enhancing plus-strand synthesis., (Copyright 2001 Academic Press.)

The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication., (Copyright 2001 Academic Press.)

Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.

Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66-73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3'-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5'-terminal 1.4 kb derived from the 5'-end of dsRNA 1 and the 3'-terminal 2.2 kb from the 3'-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A)(+). The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5'-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5'-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40-70 residues., (Copyright 2000 Academic Press.)

Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication., (Copyright 1999 Academic Press.)

In vivo genetic selection was used to study the sequences and structures required for accumulation of subviral sat-RNA C associated with turnip crinkle virus (TCV). This technique is advantageous over site-specific mutagenesis by allowing side-by-side selection from numerous sequence possibilities as well as sequence evolution. A 22 base hairpin and 6 base single-stranded tail located at the 3'-terminus of sat-RNA C were previously identified as the promoter for minus strand synthesis. Approximately 50% of plants co-inoculated with TCV and sat-RNA C containing randomized sequence in place of the 22 base hairpin accumulated sat-RNA in uninoculated leaves. The 22 base region differed in sat-RNA accumulating in all infected plants, but nearly all were predicted to fold into a hairpin structure that maintained the 6 base tail as a single-stranded sequence. Two additional rounds of sat-RNA amplification led to four sequence family 'winners', with three families containing multiple variants, indicating that evolution of these sequences was occurring in plants. Three of the four sequence family winners had the same 3 bp at the base of the stem as wild-type sat-RNA C. Two of the winners shared 15 of 22 identical bases, including the entire stem region and extending two bases into the loop. These results demonstrate the utility of the in vivo selection approach by showing that both sequence and structure contribute to a more active 3'-end region for accumulation of sat-RNA C.

The hairpin ribozyme is a self-cleaving motif found in the negatives strand of the satellite RNA of some plant viruses. In its natural context, the ribozyme comprises four helices, two of which contain conserved formally unpaired loops, that are adjacent arms of a four-way RNA junction. We show that the arms that would carry these loops are brought close together in the global conformation of the isolated junction. Using fluorescence resonance energy transfer, we demonstrate a two-magnesium ion-dependent conformational transition of the complete ribozyme that brings the loopbearing arms into close physical proximity. The ribozyme is active as a four-way junction, and the rate of cleavage may be modulated by the conformation of the four-way junction.

Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L-(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L-(+)M into the corresponding circular form C-(+)M. We transiently expressed L-(+)M wild type and L-(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C-(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L-(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53-211 and 268-350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L-(+)M. The mutant L-(+)M was found to be extensively ligated to C-(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3' terminal sequence. A trinucleotide in the 3' region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C-(+)M formation. These results suggest that the 3' stem contributes to the suitability of the small L-(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structure in vivo.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Satellite (sat-) RNA C, one of the nonessential subviral RNAs of turnip crinkle virus (TCV), is dependent on the TCV-encoded RNA-dependent RNA polymerase (RdRp) for its replication. Earlier work showed that a stem-loop structure at the 3' end of (+)-strand sat-RNA C is required for synthesis of (-)-strands in vitro using a partially purified, template-specific TCV RdRp (Song C, Simon AE, 1995, J Mol Biol 254:6-14). Cis-sequences on (-)-strands of sat-RNA C that can serve as separate promoters in vitro have now been defined. Two promoter sequences are located on (-)-strand sat-RNA C, one comprising 11 bases located near the 3' end, and the other consisting of 14 bases located 41 bases from the 5' end. Both promoter sequences contain multiple consecutive C residues followed by multiple consecutive purines and have no obvious secondary structure, suggesting that, along with hairpin structures, specific primary sequences can be recognized by the TCV RdRp. The 3'-proximal promoter sequence directed synthesis from the 3' terminus using (-)-strand templates with the natural sat-RNA 3' end (AUCCC-3'). When plasmid-derived bases were present at the 3' ends of the templates, both promoter sequences could direct the RdRp to initiate transcription internally at the multiple consecutive C residues within the promoters. This result suggests that multiple consecutive C residues are important for transcription initiation and that natural 3'-end sequences, when located at 3' termini, help the RdRp to initiate at the 3' end of the molecule.

The negative strand of the satellite RNA of tobacco ringspot virus [(-)sTRSV] is a self-cleaving RNA, of which self-cleaving domain is called the hairpin ribozyme. The negative strand of the satellite RNA of arabis mosaic virus [(-)sArMV] has been suggested to have a hairpin ribozyme-like secondary structure, and we have previously shown that this hairpin domain of (-)sArMV has ribozyme activity. Here we report characterization of the cleavage reaction of the (-)sArMV hairpin ribozyme. Mutagenesis analyses in a trans-acting system revealed, surprisingly, that the wild-type ribozyme was less active than almost all the other mutant ribozymes tested. In a cis-acting system (self-cleaving reaction), however, the reaction of the RNA containing the wild-type sequence proceeds highly efficiently. This result suggests that the inefficient cleavage of the wild-type substrate in trans-acting system may be due to low efficiency at the substrate-binding step but not at the chemical cleavage step in the reaction. We also constructed a chimeric ribozyme between the catalytic hairpin domain from (-)sArMV and the substrate-binding site from (-)sTRSV. This chimeric ribozyme had the highest activity among the trans-acting hairpin ribozymes tested.

Satellite RNA of bamboo mosaic potexvirus (satBaMV) is a linear RNA molecule which encodes a 20-kDa nonstructural protein. Sequences of seven different satBaMV isolates from bamboo hosts in three genera showed 0.7% to 7.5% base variation which spanned the whole RNA molecule. However, the putative 20-kDa open reading frame was all preserved in these isolates. The phylogenetic relationship based on the nucleotide sequence did not show particular grouping of satBaMV from the host in one genus; neither was the grouping of satBaMV evident by location of sampling. Putative secondary structures of the 3' untranslated regions showed a basic pattern with conserved hexanucleotides (ACCUAA) and polyadenylation signal (AAUAAA) located in the loop regions. Although the satBaMV-encoded 20-kDa protein is a nonstructural protein, its predicted secondary structure contains eight-stranded beta-sheets which may form "jelly-roll" structure similar to that found in capsid protein encoded by satellite virus of panicum mosaic virus.

Pea enation mosaic virus (PEMV) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two RNA species, one of which is related to the genomes of luteoviruses and the other to those of umbraviruses. In both complexes, these viral RNA components may be supplemented with satellite RNAs that are dependent on the umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. Sequence analysis identified regions of similarity between the satellites of groundnut rosette virus (GRV) and PEMV, particularly at the 5' and 3' termini and around duplicate sequence repeats present in each satellite RNA. The umbravirus GRV and the umbravirus-like PEMV RNA-2 were each able to support the replication and systemic spread of homologous and heterologous satellites. The presence of the PEMV satellite in infections with GRV had no effect on symptom expression in Nicotiana spp. or in Arachis hypogaea. Likewise, in Pisum sativum, the GRV satellite had no effect on the symptoms induced by PEMV. However, the intense yellow blotch symptoms induced in Nicotiana benthamiana by the YB3 GRV satellite in conjunction with GRV were also manifested when PEMV was the helper. Although PEMV RNA-1 was capable of supporting the encapsidation and aphid transmission of the GRV satellite, no evidence was obtained that the essential role of the GRV satellite in the aphid transmission of GRV could be supplied by the PEMV satellite. These data further strengthen the hypothesis of an evolutionary relationship between PEMV and the luteovirus-umbravirus complexes.

G + C-rich viroids proper exhibit a unique dinucleotide composition in that AU and UA are much less frequent than expected. Thus, evaluation of the dinucleotide pattern allows a quick discrimination between viroids proper and similar RNAs from plants, such as hammerhead-containing viroids, satellite RNAs, and defective interfering RNAs. In addition to sequence alignment, this method might be useful for the classification of a newly found small RNA replicon.