The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs.

As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

Identification of Host Factors Interacting with a γ-Shaped RNA Element from a Plant Virus-Associated Satellite RNA.

Previously, we identified a highly conserved, γ-shaped RNA element (γRE) from satellite RNAs of cucumber mosaic virus (CMV), and we determined γRE to be structurally required for satRNA survival and the inhibition of CMV replication. It remains unknown how γRE biologically functions. In this work, pull-down assays were used to screen candidates of host factors from Nicotiana benthamiana plants using biotin-labeled γRE as bait. Nine host factors were found to interact specifically with γRE. Then, all of these host factors were down-regulated individually in N. benthamiana plants via tobacco rattle virus-induced gene silencing and tested with infection by GFP-expressing CMV (CMV-gfp) and the isolate T1 of satRNA (sat-T1). Out of nine candidates, three host factors, namely histone H3, GTPase Ran3, and eukaryotic translation initiation factor 4A, were extremely important for infection by CMV-gfp and sat-T1. Moreover, we found that cytosolic glyceraldehyde-3-phosphate dehydrogenase 2 contributed to the replication of CMV and sat-T1, but also negatively regulated CMV 2b activity. Collectively, our work provides essential clues for uncovering the mechanism by which satRNAs inhibit CMV replication.

Satellite Noncoding RNAs (ncRNA) as Cancer Biomarkers? New Insights from FA-SAT ncRNA Molecular and Clinical Profiles in Feline Mammary Tumors.

Satellite noncoding RNAs (ncRNAs) are a new frontier of cancer biology research and biomarkers. While the knowledge on ncRNAs in human cancers is still limited, studies in other species can be informative to guide future translational research and development for cancer molecular targets and diagnostics. In this context, FA-SAT is the major satellite DNA of the cat genome, which is also present in humans, being transcribed in both species. In this study, we report new insights on FA-SAT (DNA and RNA) profile in feline mammary tumors, using disease-free tissues from the same animals as reference. We quantified the FA-SAT DNA and RNA levels (long and small transcripts) by real-time quantitative polymerase chain reaction (qPCR) and RT-qPCR. The comparison of the FA-SAT DNA and RNA levels with clinicopathological parameters revealed several associations, such as (1) the FA-SAT DNA levels' positive relation with lymphovascular invasion, (2) the FA-SAT long RNA negative correlation with Ki-67 index, and its positive association with Estrogen Receptor status, and (3) the FA-SAT small RNA level positive correlation with tumor size and skin ulceration. Also, FA-SAT long RNA is correlated with ERBB2 and c-MYC RNA levels. These data collectively suggest that FA-SAT ncRNA offers prospects as a potential cancer biomarker in cats. Further studies in humans are also needed to decipher the emerging role of ncRNAs in cancer biology and precision medicine fields. This work brings new information on the relation of FA-SAT ncRNAs with the oncogenic process, uncovering a new potential cancer biomarker.

Breaking satellite silence: human satellite II RNA expression in ovarian cancer.

Multiple cancer types demonstrate abnormal expression of repetitive RNA sequences as a form of epigenetic instability. There is growing interest in understanding the role of repetitive RNAs in cancer pathogenesis and immunogenicity and in their potential role as diagnostic or therapeutic biomarkers. In this issue of the JCI, Porter and colleagues report on satellite RNA in a subset of ovarian cancers. The authors found that high expression of human satellite (HSAT) repeats - but not other families of repeats - was associated with an immunosuppressive phenotype in ovarian cancer cell lines and tumor samples. Further induction of HSAT RNA levels in vitro, surprisingly, leads to innate immune activation, suggesting a potential therapeutic strategy. This work highlights the expanding role of repetitive RNAs in tumor biology and the need to better define specific classes of repetitive elements expressed in cancer - as well as their role in tumorigenesis, tumor immunity, and the host response to cancer.

Genetic Diversity of Tomato Black Ring Virus Satellite RNAs and Their Impact on Virus Replication.

Viral satellite RNAs (satRNAs) are small subviral particles that are associated with the genomic RNA of a helper virus (HV). Their replication, encapsidation, and movement depend on the HV. In this paper, we performed a global analysis of the satRNAs associated with different isolates of tomato black ring virus (TBRV). We checked the presence of satRNAs in 42 samples infected with TBRV, performed recombination and genetic diversity analyses, and examined the selective pressure affecting the satRNAs population. We identified 18 satRNAs in total that differed in length and the presence of point mutations. Moreover, we observed a strong effect of selection operating upon the satRNA population. We also constructed infectious cDNA clones of satRNA and examined the viral load of different TBRV isolates in the presence and absence of satRNAs, as well as the accumulation of satRNA molecules on infected plants. Our data provide evidence that the presence of satRNAs significantly affects viral load; however, the magnitude of this effect differs among viral isolates and plant hosts. We also showed a positive correlation between the number of viral genomic RNAs (gRNAs) and satRNAs for two analysed TBRV isolates.

Satellite repeat RNA expression in epithelial ovarian cancer associates with a tumor-immunosuppressive phenotype.

Aberrant expression of viral-like repeat elements is a common feature of epithelial cancers, and the substantial diversity of repeat species provides a distinct view of the cancer transcriptome. Repeatome profiling across ovarian, pancreatic, and colorectal cell lines identifies distinct clustering independent of tissue origin that is seen with coding gene analysis. Deeper analysis of ovarian cancer cell lines demonstrated that human satellite II (HSATII) satellite repeat expression was highly associated with epithelial-mesenchymal transition (EMT) and anticorrelated with IFN-response genes indicative of a more aggressive phenotype. SATII expression - and its correlation with EMT and anticorrelation with IFN-response genes - was also found in ovarian cancer RNA-Seq data and was associated with significantly shorter survival in a second independent cohort of patients with ovarian cancer. Repeat RNAs were enriched in tumor-derived extracellular vesicles capable of stimulating monocyte-derived macrophages, demonstrating a mechanism that alters the tumor microenvironment with these viral-like sequences. Targeting of HSATII with antisense locked nucleic acids stimulated IFN response and induced MHC I expression in ovarian cancer cell lines, highlighting a potential strategy of modulating the repeatome to reestablish antitumor cell immune surveillance.

Overexpression of satellite RNAs in heterochromatin induces chromosomal instability and reflects drug sensitivity in mouse cancer cells.

Overexpression of satellite RNAs in heterochromatin induces chromosomal instability (CIN) through the DNA damage response and cell cycle checkpoint activation. Although satellite RNAs may be therapeutic targets, the associated mechanisms underlying drug sensitivity are unknown. Here, we determined whether satellite RNAs reflect drug sensitivity to the topoisomerase I inhibitor camptothecin (CPT) via CIN induction. We constructed retroviral vectors expressing major satellite and control viruses, infected microsatellite stable mouse colon cancer cells (CT26) and MC38 cells harboring microsatellite instability, and assessed drug sensitivity after 48 h. Cells overexpressing satellite RNAs showed clear features of abnormal segregation, including micronuclei and anaphase bridging, and elevated levels of the DNA damage marker γH2AX relative to controls. Additionally, overexpression of satellite RNAs enhanced MC38 cell susceptibility to CPT [half-maximal inhibitory concentration: 0.814 µM (control) vs. 0.332 µM (MC38 cells with a major satellite), p = 0.003] but not that of CT26. These findings imply that MC38 cells, which are unlikely to harbor CIN, are more susceptible to CIN-induced CPT sensitivity than CT26 cells, which are characterized by CIN. Furthermore, CPT administration upregulated p53 levels but not those of p21, indicating that overexpression of major satellite transcripts likely induces CPT-responsive cell death rather than cellular senescence.

Alpha Satellite RNA Levels Are Upregulated in the Blood of Patients with Metastatic Castration-Resistant Prostate Cancer.

The aberrant overexpression of alpha satellite DNA is characteristic of many human cancers including prostate cancer; however, it is not known whether the change in the alpha satellite RNA amount occurs in the peripheral tissues of cancer patients, such as blood. Here, we analyse the level of intracellular alpha satellite RNA in the whole blood of cancer prostate patients at different stages of disease and compare it with the levels found in healthy controls. Our results reveal a significantly increased level of intracellular alpha satellite RNA in the blood of metastatic cancers patients, particularly those with metastatic castration-resistant prostate cancer relative to controls. In the blood of patients with localised tumour, no significant change relative to the controls was detected. Our results show a link between prostate cancer pathogenesis and blood intracellular alpha satellite RNA levels. We discuss the possible mechanism which could lead to the increased level of blood intracellular alpha satellite RNA at a specific metastatic stage of prostate cancer. Additionally, we analyse the clinically accepted prostate cancer biomarker PSA in all samples and discuss the possibility that alpha satellite RNA can serve as a novel prostate cancer diagnostic blood biomarker.

Proteasome inhibition alters mitotic progression through the upregulation of centromeric α-Satellite RNAs.

Cell cycle progression requires control of the abundance of several proteins and RNAs over space and time to properly transit from one phase to the next and to ensure faithful genomic inheritance in daughter cells. The proteasome, the main protein degradation system of the cell, facilitates the establishment of a proteome specific to each phase of the cell cycle. Its activity also strongly influences transcription. Here, we detected the upregulation of repetitive RNAs upon proteasome inhibition in human cancer cells using RNA-seq. The effect of proteasome inhibition on centromeres was remarkable, especially on α-Satellite RNAs. We showed that α-Satellite RNAs fluctuate along the cell cycle and interact with members of the cohesin ring, suggesting that these transcripts may take part in the regulation of mitotic progression. Next, we forced exogenous overexpression and used gapmer oligonucleotide targeting to demonstrate that α-Sat RNAs have regulatory roles in mitosis. Finally, we explored the transcriptional regulation of α-Satellite DNA. Through in silico analyses, we detected the presence of CCAAT transcription factor-binding motifs within α-Satellite centromeric arrays. Using high-resolution three-dimensional immuno-FISH and ChIP-qPCR, we showed an association between the α-Satellite upregulation and the recruitment of the transcription factor NFY-A to the centromere upon MG132-induced proteasome inhibition. Together, our results show that the proteasome controls α-Satellite RNAs associated with the regulation of mitosis.

A plant virus satellite RNA directly accelerates wing formation in its insect vector for spread.

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector.

Multiple cellular compartments engagement in Nicotiana benthamiana-peanut stunt virus-satRNA interactions revealed by systems biology approach.

KEY MESSAGE: PSV infection changed the abundance of host plant's transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and cytosol, affecting photosynthesis, translation, transcription, and splicing. Virus infection is a process resulting in numerous molecular, cellular, and physiological changes, a wide range of which can be analyzed due to development of many high-throughput techniques. Plant RNA viruses are known to replicate in the cytoplasm; however, the roles of chloroplasts and other cellular structures in the viral replication cycle and in plant antiviral defense have been recently emphasized. Therefore, the aim of this study was to analyze the small RNAs, transcripts, proteins, and phosphoproteins affected during peanut stunt virus strain P (PSV-P)-Nicotiana benthamiana interactions with or without satellite RNA (satRNA) in the context of their cellular localization or functional connections with particular cellular compartments to elucidate the compartments most affected during pathogenesis at the early stages of infection. Moreover, the processes associated with particular cell compartments were determined. The 'omic' results were subjected to comparative data analyses. Transcriptomic and small RNA (sRNA)-seq data were obtained to provide new insights into PSV-P-satRNA-plant interactions, whereas previously obtained proteomic and phosphoproteomic data were used to broaden the analysis to terms associated with cellular compartments affected by virus infection. Based on the collected results, infection with PSV-P contributed to changes in the abundance of transcripts and proteins associated with various cellular compartments, including ribosomes, chloroplasts, mitochondria, the nucleus and the cytosol, and the most affected processes were photosynthesis, translation, transcription, and mRNA splicing. Furthermore, sRNA-seq and phosphoproteomic analyses indicated that kinase regulation resulted in decreases in phosphorylation levels. The kinases were associated with the membrane, cytoplasm, and nucleus components.

The Nuclear Matrix Protein SAFB Cooperates with Major Satellite RNAs to Stabilize Heterochromatin Architecture Partially through Phase Separation.

Interphase chromatin is hierarchically organized into higher-order architectures that are essential for gene functions, yet the biomolecules that regulate these 3D architectures remain poorly understood. Here, we show that scaffold attachment factor B (SAFB), a nuclear matrix (NM)-associated protein with RNA-binding functions, modulates chromatin condensation and stabilizes heterochromatin foci in mouse cells. SAFB interacts via its R/G-rich region with heterochromatin-associated repeat transcripts such as major satellite RNAs, which promote the phase separation driven by SAFB. Depletion of SAFB leads to changes in 3D genome organization, including an increase in interchromosomal interactions adjacent to pericentromeric heterochromatin and a decrease in genomic compartmentalization, which could result from the decondensation of pericentromeric heterochromatin. Collectively, we reveal the integrated roles of NM-associated proteins and repeat RNAs in the 3D organization of heterochromatin, which may shed light on the molecular mechanisms of nuclear architecture organization.

FA-SAT ncRNA interacts with PKM2 protein: depletion of this complex induces a switch from cell proliferation to apoptosis.

FA-SAT is a highly conserved satellite DNA sequence transcribed in many Bilateria species. To disclose the cellular and functional profile of FA-SAT non-coding RNAs, a comprehensive experimental approach, including the transcripts location in the cell and in the cell cycle, the identification of its putative protein interactors, and silencing/ectopic expression phenotype analysis, was performed. FA-SAT non-coding RNAs play a nuclear function at the G1 phase of the cell cycle and the interactomic assay showed that the PKM2 protein is the main interactor. The disruption of the FA-SAT non-coding RNA/PKM2 protein complex, by the depletion of either FA-SAT or PKM2, results in the same phenotype-apoptosis, and the ectopic overexpression of FA-SAT did not affect the cell-cycle progression, but promotes the PKM2 nuclear accumulation. Overall, our data first describe the importance of this ribonucleoprotein complex in apoptosis and cell-cycle progression, what foresees a promising novel candidate molecular target for cancer therapy and diagnosis.

Two distinct nuclear stress bodies containing different sets of RNA-binding proteins are formed with HSATIII architectural noncoding RNAs upon thermal stress exposure.

Nuclear stress bodies (nSBs) are thermal stress-inducible membrane-less nuclear bodies that are formed on highly repetitive satellite III architectural noncoding RNAs (HSATIII arcRNAs). Upon thermal stress exposure, HSATIII expression is induced to sequestrate specific sets of RNA-binding proteins and form nSBs. The major population of nSBs contain SAFB as a marker, whereas the minor population are SAFB-negative. Here, we found that HNRNPM, which was previously reported to localize in nuclear foci adjacent to SAFB-positive foci upon thermal stress, localizes in a minor population of HSATIII-dependent nSBs. Hence, we used the terms nSB-S and nSB-M to distinguish the SAFB foci and HNRNPM foci, respectively. Analysis of the components of the nSBs revealed that each set contains distinct RNA-binding proteins, including SLTM and NCO5A in nSB-Ss and HNRNPA1 and HNRNPH1 in nSB-Ms. Overall, our findings indicate that two sets of nSBs containing HSATIII arcRNAs and distinct sets of RNA-binding proteins are formed upon thermal stress exposure.

Nuclear stress bodies: Interaction of its components in oncogenic regulation.

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.

Peanut Stunt Virus and Its Satellite RNA Trigger Changes in Phosphorylation in N. benthamiana Infected Plants at the Early Stage of the Infection.

Signaling in host plants is an integral part of a successful infection by pathogenic RNA viruses. Therefore, identifying early signaling events in host plants that play an important role in establishing the infection process will help our understanding of the disease process. In this context, phosphorylation constitutes one of the most important post-translational protein modifications, regulating many cellular signaling processes. In this study, we aimed to identify the processes affected by infection with Peanut stunt virus (PSV) and its satellite RNA (satRNA) in Nicotiana benthamiana at the early stage of pathogenesis. To achieve this, we performed proteome and phosphoproteome analyses on plants treated with PSV and its satRNA. The analysis of the number of differentially phosphorylated proteins showed strong down-regulation in phosphorylation in virus-treated plants (without satRNA). Moreover, proteome analysis revealed more down-regulated proteins in PSV and satRNA-treated plants, which indicated a complex dependence between proteins and their modifications. Apart from changes in photosynthesis and carbon metabolism, which are usually observed in virus-infected plants, alterations in proteins involved in RNA synthesis, transport, and turnover were observed. As a whole, this is the first community (phospho)proteome resource upon infection of N. benthamiana with a cucumovirus and its satRNA and this resource constitutes a valuable data set for future studies.

The Defense Response of Nicotiana benthamiana to Peanut Stunt Virus Infection in the Presence of Symptom Exacerbating Satellite RNA.

Peanut stunt virus (PSV) is a widespread disease infecting legumes. The PSV strains are classified into four subgroups and some are defined by the association of satellite RNAs (satRNAs). In the case of PSV, the presence of satRNAs alters the symptoms of disease in infected plants. In this study, we elucidated the plant response to PSV-G strain, which occurs in natural conditions without satRNA. However, it was found that it might easily acquire satRNA, which exacerbated pathogenesis in Nicotiana benthamiana. To explain the mechanisms underlying PSV infection and symptoms exacerbation caused by satRNA, we carried out transcriptome profiling of N. benthamiana challenged by PSV-G and satRNA using species-specific microarrays. Co-infection of plants with PSV-G + satRNA increased the number of identified differentially expressed genes (DEGs) compared with the number identified in PSV-G-infected plants. In both treatments, the majority of up-regulated DEGs were engaged in translation, ribosome biogenesis, RNA metabolism, and response to stimuli, while the down-regulated DEGs were required for photosynthesis. The presence of satRNA in PSV-G-infected plants caused different trends in expression of DEGs associated with phosphorylation, ATP binding, and plasma membrane.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells.