Satellite RNA Research Articles

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus-based gene-silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus-induced gene-silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene-silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N.benthamiana. GFP transgenic plants co-agro-inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat-shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV- but not BaMV-based vector could enhance gene-silencing efficiency in newly emerging leaves of N.benthamiana deficient in RNA-dependant RNA polymerase 6. The dual gene-silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.

A Bromodomain-Containing Host Protein Mediates the Nuclear Importation of a Satellite RNA of Cucumber Mosaic Virus

Replication of the satellite RNA (satRNA) of Cucumber Mosaic Virus is dependent on replicase proteins of helper virus (HV). However, we recently demonstrated that like with Potato spindle tuber viroid (PSTVd), a satRNA associated with Cucumber Mosaic Virus strain Q (Q-satRNA) has the propensity to localize in the nucleus and generate multimers that subsequently serve as templates for HV-dependent replication. But the mechanism regulating the nuclear importation of Q-satRNA is unknown. Here we show that the nuclear importation of Q-satRNA is mediated by a bromodomain-containing host protein (BRP1), which is also apparently involved in the nuclear localization of PSTVd. A comparative analysis of nuclear and cytoplasmic fractions from Nicotiana benthamiana plants coinfected with Q-satRNA and its HV confirmed the association of Q-satRNA but not HV with the nuclear compartment. A combination of the MS2-capsid protein-based RNA tagging assay and confocal microscopy demonstrated that the nuclear localization of Q-satRNA was completely blocked in transgenic lines of Nicotiana benthamiana (ph5.2nb) that are defective in BRP1 expression. This defect, however, was restored when the ph5.2nb lines of N. benthamiana were trans-complemented by ectopically expressed BRP1. The binding specificity of BRP1 with Q-satRNA was confirmed in vivo and in vitro by coimmunoprecipitation and electrophoretic mobility shift assays, respectively. Finally, infectivity assays involving coexpression of Q-satRNA and its HV in wild-type and ph5.2nb lines of N. benthamiana accentuated a biological role for BRP1 in the Q-satRNA infection cycle. The significance of these results in relation to a possible evolutionary relationship to viroids is discussed.

Nucleotide sequence diversity in Velvet tobacco mottle virus: a virus with a unique Australian pathosystem

Velvet tobacco mottle virus (VTMoV) is a naturally occurring mirid-transmitted sobemovirus of native velvet tobacco (Nicotiana velutina) plants in the Australian arid zone. We have sequenced the coding region of a typical field isolate of VTMoV (isolate I-17-04, satellite-plus) and show that it differed by nine polymorphisms from the previously sequenced atypical ‘satellite-minus’ variant VTMoV-K1 (represented here as L-K1-04), while retaining the same genomic and amino acid sequence motifs. We also report that although L-K1-04 was confirmed to be free of detectable satellite RNA by gel electrophoretic assay, the satellite sequence was detected in it by RT-PCR assay. Nucleotide sequence variation among the RNA-dependent RNA polymerase open reading frames of 15 field and laboratory isolates identified four phylogenetic groups, but these did not show a pattern related to site or time of sampling. This result would be consistent with nucleotide sequence variants of VTMoV being dispersed widely by migrating adult mirid vectors.

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.

Genetic Variability, Evolution, and Biological Effects of Grapevine fanleaf virus Satellite RNAs

Large satellite RNAs (type B satRNAs) of Grapevine fanleaf virus (GFLV) from the genus Nepovirus, family Secoviridae were identified in a naturally infected vineyard and a grapevine germplasm collection. These GFLV satRNA variants had a higher nucleotide sequence identity with satRNAs of Arabis mosaic virus (ArMV) strains NW and J86 (93.8 to 94.6%) than with the satRNA of GFLV strain F13 and those of other ArMV strains (68.3 to 75.0%). Phylogenetic analyses showed no distinction of GFLV and ArMV satRNAs with respect to the identity of the helper virus. Seven stretches of 8 to 15 conserved nucleotides (I-VII) were identified in the 5' region of subgroup A nepovirus genomic RNAs GFLV, ArMV, and Grapevine deformation virus) and nepovirus type B satRNAs, including previously reported motif I, suggesting that large satRNAs might have originated from recombination between an ancestral subgroup A nepovirus RNA and an unknown RNA sequence with the 5' region acting as a putative cis-replication element. A comparative analysis of two GFLV strains carrying or absent of satRNAs showed no discernable effect on virus accumulation and symptom expression in Chenopodium quinoa, a systemic herbaceous host. This work sheds light on the origin and biological effects of large satRNAs associated with subgroup A nepoviruses.

Viroid-like RNAs from cherry trees affected by leaf scorch disease: further data supporting their association with mycoviral double-stranded RNAs

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88%), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

Complete Nucleotide Sequence of a South African Isolate of Grapevine Fanleaf Virus and Its Associated Satellite RNA

The complete sequences of RNA1, RNA2 and satellite RNA have been determined for a South African isolate of Grapevine fanleaf virus (GFLV-SACH44). The two RNAs of GFLV-SACH44 are 7,341 nucleotides (nt) and 3,816 nt in length, respectively, and its satellite RNA (satRNA) is 1,104 nt in length, all excluding the poly(A) tail. Multiple sequence alignment of these sequences showed that GFLV-SACH44 RNA1 and RNA2 were the closest to the South African isolate, GFLV-SAPCS3 (98.2% and 98.6% nt identity, respectively), followed by the French isolate, GFLV-F13 (87.3% and 90.1% nt identity, respectively). Interestingly, the GFLV-SACH44 satRNA is more similar to three Arabis mosaic virus satRNAs (85%–87.4% nt identity) than to the satRNA of GFLV-F13 (81.8% nt identity) and was most distantly related to the satRNA of GFLV-R2 (71.0% nt identity). Full-length infectious clones of GFLV-SACH44 satRNA were constructed. The infectivity of the clones was tested with three nepovirus isolates, GFLV-NW, Arabis mosaic virus (ArMV)-NW and GFLV-SAPCS3. The clones were mechanically inoculated in Chenopodium quinoa and were infectious when co-inoculated with the two GFLV helper viruses, but not when co-inoculated with ArMV-NW.

A novel dsRNA element isolated from the Aspergillus foetidus mycovirus complex

Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes.

The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by Peanut stunt virus in the presence or absence of its satellite RNA

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts (GTs) with and without PSV-P satRNA and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin-NADP-reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60β--whose abundance of the proteins changed for all comparisons--and aminopeptidase that is a satRNA- or PSV-P-GT/satRNA-responsive protein. Additionally, the levels of the stress-related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV-P-GT- and PSV-P-GT/satRNA-infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

In Silico Analysis the Complementarity of Tomato MicroRNA/MicroRNA* Sequences with Cucumber Mosaic Virus (CMV) Genomic RNAs

Usually, modifications in normal microRNAs (miRs)-dependent gene expression are induced in transgenic plants expressing viral silencing suppressors or in viruses-inoculated plants. These phenomena are generally interpreted as a side effect of the antiviral silencing suppression. However, it has been recently reported that certain animal miRs prevent viral infection by directly interfering with pathogen replication or by binding to viral transcripts. To investigate whether such mechanisms operate in plants, we performed in silico analysis using tomato miR/miR* sequences and genomes of Cucumber mosaic virus (CMV)-Fny (the severe strain), CMV-Q (the mild strain), the aggravated satellite RNA (satRNA) variant satT1 and the attenuated variant satYn12. Results showed that a total of 38 and 37 miR/miR* sequences could bind to genomic RNAs of CMV-Fny and CMV-Q, respectively. But only one sequence could bind to safT1 and three to satYn12. Interestingly, we found that these miRs/miR*s exhibited significant complementarity within 2a and 3a Open Reading Frames (ORFs) of CMV. And most of the sequences with potential to bind viral genomes were of miR*s, suggesting novel role of miR*s in host defense response. This finding is noteworthy as plant miR* sequences are presently thought very unstable and are not assigned with any function. Finally, we could predict targets in tomato database for the miR/miR* sequences that are highlighted in our study, most of them are involved in plant metabolism. Overall, these results shed light on the possible novel role of plant miR/miR* in antiviral defense mechanisms.

L-A-lus, a New Variant of the L-A Totivirus Found in Wine Yeasts with Klus Killer Toxin-Encoding Mlus Double-Stranded RNA: Possible Role of Killer Toxin-Encoding Satellite RNAs in the Evolution of Their Helper Viruses

Yeast killer viruses are widely distributed in nature. Several toxins encoded in double-stranded RNA (dsRNA) satellites of the L-A totivirus have been described, including K1, K2, K28, and Klus. The 4.6-kb L-A genome encodes the Gag major structural protein that forms a 39-nm icosahedral virion and Gag-Pol, a minor fusion protein. Gag-Pol has transcriptase and replicase activities responsible for maintenance of L-A (or its satellite RNAs). Recently we reported a new killer toxin, Klus. The L-A virus in Klus strains showed poor hybridization to known L-A probes, suggesting substantial differences in their sequences. Here we report the characterization of this new L-A variant named L-A-lus. At the nucleotide level, L-A and L-A-lus showed only 73% identity, a value that increases to 86% in the amino acid composition of Gag or Gag-Pol. Two regions in their genomes, however, the frameshifting region between Gag and Pol and the encapsidation signal, are 100% identical, implying the importance of these two cis signals in the virus life cycle. L-A-lus shows higher resistance than L-A to growth at high temperature or to in vivo expression of endo- or exonucleases. L-A-lus also has wider helper activity, being able to maintain not only Mlus but also M1 or a satellite RNA of L-A called X. In a screening of 31 wine strains, we found that none of them had L-A; they carried either L-A-lus or a different L-A variant in K2 strains. Our data show that distinct M killer viruses are specifically associated with L-As with different nucleotide compositions, suggesting coevolution.

Transgenic resistance to Bamboo mosaic virus by expression of interfering satellite RNA

Plant genetic engineering has broadened the options for plant virus resistance and is mostly based on pathogen-derived resistance. Previously, we have shown that interfering satellite RNA (satRNA) of Bamboo mosaic virus (satBaMV) greatly reduces Bamboo mosaic virus (BaMV) accumulation and BaMV-induced symptoms in co-inoculated plants. Here, we generated a nonviral source of virus-resistant transgenic Nicotiana benthamiana and Arabidopsis thaliana by introducing interfering satBaMV. Asymptomatic transgenic N. benthamiana lines were highly resistant to BaMV virion and viral RNA infection, and the expression of the transgene BSL6 was higher in asymptomatic than mildly symptomatic lines. In addition, BaMV- and satBaMV-specific small RNAs were detectable only after BaMV challenge, and their levels were associated with genomic viral RNA or satRNA levels. By transcriptomic analysis, the salicylic acid (SA) signalling pathway was not induced in satBaMV transgenic A. thaliana in mock conditions, suggesting that two major antiviral mechanisms, RNA silencing and SA-mediated resistance, are not involved directly in transgenic satBaMV-mediated BaMV interference. In contrast, resistance is associated with the level of the interfering satBaMV transgene. We propose satBaMV-mediated BaMV interference in transgenic plants by competition for replicase with BaMV.

Abstract 5291: Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis .

Abstract It was believed that most satellite repeats in pericentric heterochromatin region were silenced and rarely transcribed into RNAs. However, a few reports suggest that non-coding satellite RNAs may play a significant role in cell division, heterochromatin remodeling or DNA damage such as other well-researched non-coding RNAs. In fact, a next generation digital gene expression method recently revealed that human satellite II (HSATII) transcripts are highly and specifically upregulated in human pancreatic tumors. To further explore these findings, we examined the precise localization of HSATII transcripts in human clinical pancreatic tissues by in situ hybridization using highly sensitive locked nucleic acid-modified oligonucleotide probes. Unexpectedly, HSATII RNAs were not detected in pancreatic ductal cancerous lesions, in almost all cases examined. Instead, HSATII RNAs were clearly expressed in non-cancerous adjacent acinar cells, surrounding carcinoma tissues. On the other hand, none of normal or inflammatory pancreatic tissues derived from the ones without cancer expressed HSATII RNAs. While, HSATII transcript expression in normal pancreatic ductal cells could not be induced in vitro even under stress conditions which mimic the tumor-adjacent environments, the cells with ectopic expression of HSATII transcripts showed the increase foci of gH2AX, which may reflect the increased genomic instability. These results suggest that the expression of HSATII may not be phenotypic changes in cancers but may directly contribute to the oncogenesis in the cells in precancerous lesions. Citation Format: Takahiro Kishikawa. Non-coding satellite RNAs are highly expressed in cancer adjacent lesions of human pancreatic carcinoma and may promote oncogenesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5291. doi:10.1158/1538-7445.AM2013-5291

VIRAL SATELLITE RNA AS A BIOLOGICAL CONTROL AGENT FOR PLANTS

Biological control technology based on viral satellite mediated protection is a novel concept of the viral disease control strategies. Cucumber mosaic virus (CMV) is considered one of the most economically damaging viruses among field grown vegetables such as tomato (Solanum lycopersicum L). CMV can harbor molecular parasites known as satellite RNAs that dramatically alter the symptom phenotype induced by the virus. Two indigenous strains of CMV satellite RNA were isolated and have been sequenced. One of these strains designated as CMV‐KU1 reduced the viral disease to a virtually asymptomatic condition. The other strain was found to aggravate the symptoms and cause severe crop loss and tomato necrosis. Thus, satellites have been referred to as “natural inhibitors of crop damaging viruses’. These satellites RNAs are small nucleic acids whose nucleotide sequences are unrelated to, but are replicatively dependent upon the viral genome; they have a molecular parasitic relationship. Particularly, the KU1 strain may be used to protect plants against a particular viral strain of CMV, referred to as the KU2 strain. The attenuating properties of some satellite RNAs can be exploited to control viral diseases by preinoculating crops with an attenuating satellite RNA variant combined with virus. This technique can effectively protect against CMV disease in the field.

Analysis of the Replication Proteins and Satellites of the Turnip Crinkle Virus

The Turnip Crinkle Virus (TCV) is a single‐stranded, positivesense RNA plant virus that serves as a model organism for related human and animal RNA viruses, such as the West Nile, SARS‐CoV and Hepatitis C viruses. All positive‐sense RNA viruses replicate through asymmetrical replication; understanding the detailed biochemistry of this process should lead to potential drug targets for intervening in RNA viral infections.TCV provides an attractive model because satellite RNAs can accompany TCV infection. These satellite RNAs are minimal replicons that contain all cis‐acting RNA sequences required for replication. The goal of this research is to develop a model of replication of TCV satellites in the non‐host Saccharomyces cerevisiae, baker's yeast. Yeast provides numerous biochemical and genetic techniques not available in turnips.The key RNA replication proteins are p28 and p88. Their genes have been cloned into yeast shuttle vectors for expression in yeast and E. coli. The membrane binding protein, p28, has been visualized at its target membrane in yeast using fluorescence microscopy. Two natural satellites and a chimera of these two have also been cloned. The satellite constructs are bounded by self‐cleaving ribozymes that should generate active ends in vivo and in vitro for the replication system. An in vitro system for RNA recognition and eventually full replication will be established.

Expression of the repeat genome and aberrant epigenetic factors in cancer

Nearly half of the human genome consists of noncoding repetitive DNA elements, including tandem satellite repeats in large blocks at the pericentric regions of chromosomes and intergenic repetitive elements. While both repeat types were long thought to remain mostly silent, recent evidence indicates that repeats can be expressed, but the extent and regulation of their expression or their potential function(s) remain to be elucidated. Due to their critical location within regions vital for cell division, it is expected that tight regulation of pericentric satellite sequences is essential for both epigenetic and genetic stability. Our data suggests aberrant expression of pericentric satellite RNA is tightly linked to epigenetic misregulation in cancer. It is well known that epigenetic changes can be important in cancer initiation and progression, but studies have focused primarily on the inappropriate silencing and methylation of tumor suppressor genes. While pathologists have long noted the loss of heterochromatic organization in cancer nuclei, and hypomethylation of satellite DNA has been observed, the misregulation of repeat RNAs has only recently been described. Our results provide a link between overexpression of repeat RNAs and aberrant distribution of epigenetic factors in cancer. Our data suggests regulation of the repeat genome has potentially important roles in both normal and neoplastic cells in their ability to affect distribution and recruitment of epigenetic factors.

Current Challenges in Bacterial Transcriptomics

Over the past decade or so, dramatic developments in our ability to experimentally determine the content and function of genomes have taken place. In particular, next-generation sequencing technologies are now inspiring a new understanding of bacterial transcriptomes on a global scale. In bacterial cells, whole-transcriptome studies have not received attention, owing to the general view that bacterial genomes are simple. However, several recent RNA sequencing results are revealing unexpected levels of complexity in bacterial transcriptomes, indicating that the transcribed regions of genomes are much larger and complex than previously anticipated. In particular, these data show a wide array of small RNAs, antisense RNAs, and alternative transcripts. Here, we review how current transcriptomics are now revolutionizing our understanding of the complexity and regulation of bacterial transcriptomes.

Effects of physical exercise on the activation and differentiation potential of satellite cells from skeletal muscles of old mice: an in vitro study.

Sarcopenia is an age-related loss of muscle mass, strength and function and represents an important risk factor for physical disability in elderly (Thompson, 2009). The mechanisms leading to sarcopenia are still largely unknown and no specific therapy is at present available to counteract its onset or progress. Many studies have stressed the importance of physical exercise as an effective approach to prevent/limit the sarcopenic drive (Zancanaro et al., 2007; Malatesta et al., 2011). The aim of this investigation was to evaluate the effects of adapted physical exercise (treadmill running) on the proliferation and differentiation potential of cultured satellite-cell-derived myoblasts isolated from skeletal muscles of old mice. Ultrastructural morphology and immunocytochemistry were applied, with special attention to molecular markers of satellite cell activation and RNA processing. The results demonstrate that treadmill running markedly improved the structural and functional features of both myoblasts and myotubes in comparison to old sedentary mice.

Hepatitis Delta Virus: A Peculiar Virus

The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology.