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Exoribonuclease R (RNase R) is a 3′ hydrolytic exoribonuclease that can degrade structured RNA. Mutation in RNase R affects virulence of certain human pathogenic bacteria. The aim of this study was to determine whether RNase R is necessary for virulence of the phytopathogen that causes bacterial blight in rice, Xanthomonas oryzae pv. oryzae (Xoo). In silico analysis has indicated that RNase R is highly conserved among various xanthomonads. Amino acid sequence alignment of Xoo RNase R with RNase R from various taxa indicated that Xoo RNase R clustered with RNase R of order Xanthomonadales. To study its role in virulence, we generated a gene disruption mutant of Xoo RNase R. The Xoo rnr– mutant is moderately virulence deficient, and the complementing strain (rnr–/pHM1::rnr) rescued the virulence deficiency of the mutant. We investigated swimming and swarming motilities in both nutrient-deficient minimal media and nutrient-optimal media. We observed that RNase R mutation has adversely affected the swimming and swarming motilities of Xoo in optimal media. However, in nutrient-deficient media only swimming motility was noticeably affected. Growth curves in optimal media at suboptimal temperature (15°C cold stress) indicate that the Xoo rnr– mutant grows more slowly than the Xoo wild type and complementing strain (rnr–/pHM1::rnr). Given these findings, we report for the first time that RNase R function is necessary for complete virulence of Xoo in rice. It is also important for motility of Xoo in media and for growth of Xoo at suboptimal temperature.

Bacteria often encounter temperature fluctuations in their natural habitats and must adapt to survive. The molecular response of bacteria to sudden temperature upshift or downshift is termed the heat shock response (HSR) or the cold shock response (CSR), respectively. Unlike the HSR, which activates a dedicated transcription factor that predominantly copes with heat-induced protein folding stress, the CSR is mediated by a diverse set of inputs. This review provides a picture of our current understanding of the CSR across bacteria. The fundamental aspects of CSR involved in sensing and adapting to temperature drop, including regulation of membrane fluidity, protein folding, DNA topology, RNA metabolism, and protein translation, are discussed. Special emphasis is placed on recent findings of a CSR circuitry in Escherichia coli mediated by cold shock family proteins and RNase R that monitors and modulates messenger RNA structure to facilitate global translation recovery during acclimation.

Numerous indirect and in silico produced evidences suggest circular RNAs (circRNA) in mammals while thorough experimental proofs of their existence have rarely been reported. Biological studies of circRNA, however, should be based on experimentally verified circRNAs. Here, we describe the identification of two circRNAs originating from the gene locus of the translocation associated membrane protein 1 (TRAM1). Linear and potentially circular TRAM1-specific transcripts were identified in a transcriptome analysis of urine RNA of bladder cancer (BCa) patients versus healthy donors. Thus, we first focused on the topology of TRAM1-specific transcripts. We describe conclusive experimental evidence for the existence of TRAM1-specific circRNAs in the human BCa cell lines ECV-304 and RT-4. PCR-based methodology followed by cloning and sequencing strongly indicated the circular topology of two TRAM1 RNAs. Further, studies with exon fusion sequence-specific antisense oligonucleotides (asON) and RNase H as well as studies in the use of RNase R contribute to conclusive set of experiments supporting the circular topology of TRAM1 transcripts. On the biological side, TRAM1-specific circRNAs showed low expression levels and minor differences in BCa cell lines while linear TRAM1 transcripts displayed down-regulated expression in the higher cancer stage model ECV-304 versus more differentiated RT-4 cells.

The present work carried out in the course of this PhD is focused on the most important mechanism involved in the quality control of bacterial protein synthesis, called trans-translation. trans-translation is essential for bacteria cell survival as it allows the rescue of stalled ribosomes on non-stop mRNA during the translation, enabling their recycling with simultaneous degradation of the defective messenger and degradation of the non-functional nascent polypeptide chain. The two main players involved in this process are the transfer-messenger RNA (tmRNA), a chimeric RNA molecule capable of playing the role of both a tRNA and an mRNA, and its partner the small basic protein B (SmpB), which is essential for the ribosome binding, the correct positioning of the resume codon and the expulsion of non-stop mRNA from the ribosome. In the first section of this manuscript, we explain the structural study, by cryo- EM, of the trans-translation process that allowed us to reconstruct four high- resolution atomic structures of the ribosome in four different consecutive steps of this mechanism (pre-accommodation, accommodation, translocation and post-translocation). In this way, we were able to highlight the molecular interactions at the atomic level that are essential to the whole process and are of particular interest since they can be used as targets against which new molecules with antibacterial activity can be developed.In the second part of the work we focused our attention on the structural characterisation of a third component involved in trans-translation, known as RNase R. The RNase R is an ribonuclease essential for the degradation of non-stop mRNA. It has been observed to compete for the same binding site on the ribosome with tmRNA, and to be highly dynamic when interacting with the ribosome itself. For this reason, the study was extended to a more complex system in which we sought to understand the exact role of all three players (tmRNA, SmpB and RNase R) during the stalling of two ribosomes on the same defective mRNA. This study is still ongoing. In the last chapter, we described a particular new conformation for one of the most important proteins of the ribosome, known as bS1. This protein is essential in the early stages of translation. In this case, one of its six domains, normally known to be involved in the interaction with the ribosome, shows a particular conformation that allows it to interact with the Shine-Dalgarno portion of the mRNA, stabilising its binding to its counterpart at the 3' end of the 16S rRNA. This observation may open the door to a better understanding of the mechanism of action of this protein.; Le présent travail effectué au cours de cette thèse se concentre sur le mécanisme le plus important impliqué dans le contrôle de la qualité de la synthèse des protéines bactériennes, appelé trans-traduction. La trans- traduction est essentielle pour la survie des cellules bactériennes car elle permet le sauvetage des ribosomes bloqués sur l'ARNm non-stop pendant la traduction, permettant leur recyclage avec la dégradation simultanée du messager défectueux et la dégradation de la chaîne polypeptidique naissante non fonctionnelle. Les deux principaux acteurs impliqués dans ce processus sont l'ARN transfert-messager (ARNtm), une molécule d'ARN chimérique capable de jouer à la fois le rôle d'un ARNt et d'un ARNm, et son partenaire la petite protéine basique B (SmpB), qui est essentielle pour la liaison au ribosome, le positionnement correct du codon de reprise et l'expulsion de l'ARNm non-stop du ribosome. Dans a première section de ce manuscrit, nous expliquons l'étude structurale, par cryo-EM, du processus de trans-traduction qui nous a permis de reconstruire quatre structures atomiques à haute résolution du ribosome dans quatre différentes étapes consécutives de ce mécanisme (pré- accommodation, accommodation, translocation et post-translocation). De cette façon, nous avons pu mettre en évidence les interactions moléculaires au niveau atomique qui sont essentielles à l'ensemble du processus et qui sont particulièrement intéressantes car elles peuvent être utilisées comme cibles contre lesquelles de nouvelles molécules à activité antibactérienne peuvent être développées. Dans la deuxième partie du travail, nous avons concentré notre attention sur la caractérisation structurelle d'un troisième composant impliqué dans la trans-traduction, connu sous le nom de RNase R. La RNase R est une ribonucléase essentielle pour la dégradation de l'ARNm non-stop. Il a été observé qu'elle entre en compétition avec l'ARNtm pour le même site de liaison sur le ribosome, et qu'elle est très dynamique lorsqu'elle interagit avec le ribosome lui-même. Pour cette raison, l'étude a été étendue à un système plus complexe dans lequel nous avons cherché à comprendre le rôle exact des trois acteurs (ARNtm, SmpB et RNase R) lors du décrochage de deux ribosomes sur le même ARNm défectueux (disomes). Cette étude est toujours en cours. Dans le dernier chapitre, nous avons décrit une nouvelle conformation particulière pour l'une des protéines les plus importantes du ribosome, connue sous le nom de bS1. Cette protéine est essentielle dans les premières étapes de la traduction. Dans ce cas, l'un de ses six domaines, normalement connu pour être impliqué dans l'interaction avec le ribosome, présente une conformation particulière qui lui permet d'interagir avec la portion Shine-Dalgarno de l'ARNm, stabilisant ainsi sa liaison avec son homologue à l'extrémité 3' de l'ARNr 16S. Cette observation pourrait ouvrir la porte à une meilleure compréhension du mécanisme d'action de cette protéine.

Circular RNA (circRNA) is a large class of covalently closed circRNA. As a member of competitive endogenous RNA, it participates in the regulation of circRNA-miRNA-mRNA network and plays an important role in the regulation of physiology and pathology. CircRNA is produced by the reverse splicing of exon, intron or both, forming exon or intron circRNA. Studies have shown that circRNA is a ubiquitous molecule, which exceeds the linear mRNA distributed in human cells. Because of its covalent closed-loop structure, circRNA is resistant to RNase R, which is more stable than linear mRNA; circRNA is highly conserved in different species. It was found that circRNA competitively adsorbs miRNA, as a miRNA sponge, to involve in the expression regulation of a variety of genes and plays an important role in tumor development, invasion, metastasis and other processes. These molecules offer new potential opportunities for therapeutic intervention and serve as biomarkers for diagnosis. In this paper, the origin, characteristics and functions of circRNA and its role in tumor development, invasion and metastasis, diagnosis and prognosis are reviewed.

In self-incompatible Solanaceous species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific non-self S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Here, we first show that Petunia hybrida (Ph) S3-RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, II and III. R I is ubiquitinated in unpollinated, self- and cross-pollinated pistils, indicating its occurrence prior to PhS3-RNase uptake into pollen tubes, whereas R II and III are exclusively ubiquitinated in cross-pollinated pistils. Second, removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and III in less extents, indicating their increased cytotoxicity. In consistent, the mutated R II of PhS3-RNase resulted in marked reduction of its degradation, whereas that of R I and III in less reductions. Taken together, our results demonstrate that PhS3-RNase R II functions as a major ubiquitination region for its destruction and R I and III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.ONE SENTENCE SUMMARYBiochemical and transgenic analyses reveal that Petunia hybrida S3-RNase cytotoxicity is largely restricted by a stepwise ubiquitination and degradation pathway during cross-pollination.

RNA 2′-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3′-5′ exoribonuclease and is particularly sensitive to RNA 2′-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2′-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR’s ribonuclease activity and significant sensitivity to RNA 2′-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2′-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2′-O-methylations research in biological and clinical samples.

Ribosome hibernation is a universal translation stress response found in bacteria as well as plant plastids. The term was coined almost two decades ago and despite recent insights including detailed cryo-EM structures, the physiological role and underlying molecular mechanism of ribosome hibernation has remained unclear. Here, we demonstrate that Escherichia coli hibernation factors RMF, HPF and RaiA (HFs) concurrently confer ribosome hibernation. In response to carbon starvation and resulting growth arrest, we observe that HFs protect ribosomes at the initial stage of starvation. Consistently, a deletion mutant lacking all three factors (ΔHF) is severely inhibited in regrowth from starvation. ΔHF cells increasingly accumulate 70S ribosomes harbouring fragmented rRNA, while rRNA in wild-type 100S dimers is intact. RNA fragmentation is observed to specifically occur at HF-associated sites in 16S rRNA of assembled 70S ribosomes. Surprisingly, degradation of the 16S rRNA 3′-end is decreased in cells lacking conserved endoribonuclease YbeY and exoribonuclease RNase R suggesting that HFs directly block these ribonucleases from accessing target sites in the ribosome.

The role of archetypal ribonucleases (RNases) in the physiology and stress endurance of the soil bacterium and metabolic engineering platform Pseudomonas putida KT2440 has been inspected. To this end, variants of this strain lacking each of the most important RNases were constructed. Each mutant lacked either one exoribonuclease (PNPase, RNase R) or one endoribonuclease (RNase E, RNase III, RNase G). The global physiological and metabolic costs of the absence of each of these enzymes were then analysed in terms of growth, motility and morphology. The effects of different oxidative chemicals that mimic the stresses endured by this microorganism in its natural habitats were studied as well. The results highlighted that each ribonuclease is specifically related with different traits of the environmental lifestyle that distinctively characterizes this microorganism. Interestingly, the physiological responses of P. putida to the absence of each enzyme diverged significantly from those known previously in Escherichia coli. This exposed not only species-specific regulatory functions for otherwise known RNase activities but also expanded the panoply of post-transcriptional adaptation devices that P. putida can make use of for facing hostile environments.

Introduction Owing to its involvement in both the initiation and progression of various cancers, aberrant circular RNA (circRNA) expression has been researched extensively in the recent times. In the present study, we aim to investigate the effect of a novel circRNA has_circ_0025933 (circNELL2) in the progression of esophageal squamous cell carcinoma (ESCC). Materials and methods Sanger sequencing and the detection of circNELL2 level after RNase R or actinomycin D treatment were performed to identify the existence of cirNELL2 in ESCC cells. WST, EDU staining and colony-formation assay were used to assess the proliferation while transwell assay was used to evaluate the migration of ESCC cells. Luciferase assay, RNA pull down and the FISH assay were performed to verify the interaction between circNELL2 and miR-127-5p as well as miR-127-5p and CDC6. Xenograft model was carried out to evaluate the effect of circNELL2 in vivo. Results circNELL2 was proved to exist in ESCC cells. The up-regulated expression of circNELL2 in the clinical ESCC specimens was also verified. Next, function studies suggested that circNELL2 knockdown inhibited the proliferation of ESCC cells in vitro and in vivo, while circNELL2 overexpression promotes that of ESCC cells. Besides, this study mechanically predicted and verified the target miR of circNELL2, which is miR-127-5p. It was found that miR-127-5p was capable of reversing the effect of circNELL2 on ESCC cells. Moreover, miR-127-5p was also found to target CDC6 to participate in the regulation of cell phenotype. Discussion circNELL2 promoted the progression of ESCC cells via sponging miR-127-5p, and it has the potential to serve as a novel prognostic and therapeutic target for ESCC.

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure–function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule–RNA targeting.

Motivation The ubiquitous abundance of circular RNAs (circRNAs) has been revealed by performing high-throughput sequencing in a variety of eukaryotes. circRNAs are related to some diseases, such as cancer in which they act as oncogenes or tumor-suppressors and, therefore, have the potential to be used as biomarkers or therapeutic targets. Accurate and rapid detection of circRNAs from short reads remains computationally challenging. This is due to the fact that identifying chimeric reads, which is essential for finding back-splice junctions, is a complex process. The sensitivity of discovery methods, to a high degree, relies on the underlying mapper that is used for finding chimeric reads. Furthermore, all the available circRNA discovery pipelines are resource intensive. Results We introduce CircMiner, a novel stand-alone circRNA detection method that rapidly identifies and filters out linear RNA sequencing reads and detects back-splice junctions. CircMiner employs a rapid pseudo-alignment technique to identify linear reads that originate from transcripts, genes or the genome. CircMiner further processes the remaining reads to identify the back-splice junctions and detect circRNAs with single-nucleotide resolution. We evaluated the efficacy of CircMiner using simulated datasets generated from known back-splice junctions and showed that CircMiner has superior accuracy and speed compared to the existing circRNA detection tools. Additionally, on two RNase R treated cell line datasets, CircMiner was able to detect most of consistent, high confidence circRNAs compared to untreated samples of the same cell line. Availability and implementation CircMiner is implemented in C++ and is available online at https://github.com/vpc-ccg/circminer. Supplementary information Supplementary data are available at Bioinformatics online.

Background Recently, many emerging circular RNAs (circRNAs) have been studied in human malignancies, including gastric cancer (GC). Researches concerning cancers have revealed that aberrant expression of circRNAs play a big part in tumorigenesis and development of diverse malignant tumors. Although hsa_circ_0014130 (circPIP5K1A) has been confirmed to be closely related to non-small cell lung cancer (NSCLC) progression, the knowledge of its function on GC progression remains unclear. Therefore, it is of great interest to uncover the underlying role of circPIP5K1A in GC. Methods The expression and characteristic of circPIP5K1A were separately analyzed by RT-qPCR, nucleic acid electrophoresis, RNase R and Actinomycin D treatment. CCK-8, colony formation, EdU, transwell, TUNEL, flow cytometry, luciferase reporter, RIP and RNA pull-down assays were employed to testify the regulatory role of circPIP5K1A in GC. Results In current study, circPIP5K1A, featured with closed-loop structure, was proved to be highly expressed in tissues and cells of GC. Loss-of-function assays depicted that silencing circPIP5K1A suppressed GC development. Follow-up mechanism tests unveiled that circPIP5K1A bound with miR-376c-3p and inhibition of miR-376c-3p reversed circPIP5K1A downregulation-mediated effect on GC progression. Additionally, ZNF146 was verified to be the downstream molecule of circPIP5K1A/miR-376c-3p axis in modulating GC progression. Conclusions circPIP5K1A stimulates GC progression by sponging miR-376c-3p to upregulate ZNF146 expression.

Circular RNAs (circRNAs) are generally formed by back splicing and are expressed in various cells. Vascular calcification (VC), a common complication of chronic kidney disease (CKD), is often associated with cardiovascular disease. The relationship between circRNAs and VC has not yet been studied. Inorganic phosphate (Pi) was used to treat rat vascular smooth muscle cells to induce VC. circRNAs were identified by analyzing RNA sequencing (RNA-seq) data, and their expression change during VC was validated. The selected circRNAs, including circSamd4a, circSmoc1-1, circMettl9, and circUxs1, were resistant to RNase R digestion and mostly localized in the cytoplasm. While silencing circSamd4a promoted VC, overexpressing it reduced VC in calcium assay and Alizarin red S (ARS) staining. In addition, microRNA (miRNA) microarray, luciferase reporter assay, and calcium assay suggested that circSamd4a could act as a miRNA suppressor. Our data show that circSamd4a has an anti-calcification role by functioning as a miRNA sponge. Moreover, mRNAs that can interact with miRNAs were predicted from RNA-seq and bioinformatics analysis, and the circSamd4a-miRNA-mRNA axis involved in VC was verified by luciferase reporter assay and calcium assay. Since circSamd4a is conserved in humans, it can serve as a novel therapeutic target in resolving VC.

Background To investigate the effect of circular RNA circ ACAP2 on apoptotic phenotype of rat cardiomyocytes and its molecular mechanism. Methods Left anterior descending ligation was used to establish a rat myocardial infarction (MI) model, and real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of circ ACAP2 in apoptotic rat cardiomyocytes. The stability of circ ACAP2 was tested by actinomycin D and RNase R. H9C2 cells were infected by recombinant circ ACAP2 adenovirus (rAd-circ ACAP2), and the expression of apoptotic related genes Bax and BCL-2 in H9C2 was detected. Double luciferase reporter gene experiment, RNA antisense purification experiment and RNA Pull-Down experiments verified the binding effect of circ ACAP2 and miR-29. Results RT-qPCR results showed that the expression of circ ACAP2 was increased in cardiomyocytes of MI rats. Actinomycin D and RNase R experiment confirmed that circ ACAP2 had better stability and resistance to RNase R degradation than its host gene ACAP2 mRNA. Overexpression of circ ACAP2 promotes apoptosis. The double luciferase reporter gene assay, RNA antisense purification assay and RNA Pulldown assay consistently confirmed the specific binding effect between circ ACAP2 and miR-29, and circ ACAP2 promoted the apoptotic phenotype in rats. Conclusions The expression of circ ACAP2 increased in cardiomyocytes after MI and promoted apoptosis by binding to miR-29.

Simple Summary Circular RNAs are a recently appreciated class of regulatory non-coding RNAs. Although a number of high-resolution methods have been developed for the imaging of RNAs in cells and tissues, there is no reliable method for the simultaneous imaging of circular RNAs distinctly from their linear counterparts. Here, we report circFISH as a simple and single-molecule resolution method that will simultaneously image linear and circular RNAs in fixed cells and tissues. We demonstrate that multiple circular RNAs can be imaged using circFISH. We also show the ability of circFISH to work seamlessly with protein and other organelle imaging. We optimized the method to work across sample types, making it a versatile tool for the functional characterization of circular RNAs. Abstract Circular RNAs (circRNAs) are regulatory RNAs which have recently been shown to have clinical significance in several diseases, including, but not limited to, various cancers, neurological diseases and cardiovascular diseases. The function of such regulatory RNAs is largely dependent on their subcellular localization. Several circRNAs have been shown to conduct antagonistic roles compared to the products of the linear isoforms, and thus need to be characterized distinctly from the linear RNAs. However, conventional fluorescent in situ hybridization (FISH) techniques cannot be employed directly to distinguish the signals from linear and circular isoforms because most circRNAs share the same sequence with the linear RNAs. In order to address this unmet need, we adapted the well-established method of single-molecule FISH by designing two sets of probes to differentiate the linear and circular RNA isoforms by virtue of signal colocalization. We call this method ‘circular fluorescent in situ hybridization’ (circFISH). Linear and circular RNAs were successfully visualized and quantified at a single-molecule resolution in fixed cells. RNase R treatment during the circFISH reduced the levels of linear RNAs while the circRNA levels remain unaltered. Furthermore, cells with shRNAs specific to circRNA showed the loss of circRNA levels, whereas the linear RNA levels were unaffected. The optimization of the in-situ RNase R treatment allowed the multiplexing of circFISH to combine it with organelle staining. CircFISH was found to be compatible with multiple sample types, including cultured cells and fresh-frozen and formalin-fixed tissue sections. Thus, we present circFISH as a versatile method for the simultaneous visualization and quantification of the distribution and localization of linear and circular RNA in fixed cells and tissue samples.

Some sea cucumbers are economically and ecologically important, but they are threatened by thermal and hypoxic stress in changing oceanographic conditions. We construct circRNAs profiles, reveal circRNAs characters, and illustrate the potential regulatory roles of circRNAs in one commercially important species of sea cucumber, Apostichopus japonicus. Reads are distributed in intergenic (44.14%), exonic (48.26%) and intronic (7.60%) regions of the genome. A total of 1684 circRNAs were identified, and the most common spliced length is 269 nt in the present study. In three treatments (HT [thermal stress], LO [hypoxic stress], and HL [combined thermal and hypoxic stress]), 24, 27 and 27 differentially expressed (DE) circRNAs were identified, respectively. Five novel DE-circRNAs commonly occur in these treatments (novel_circ_0003311, novel_circ_0000229, novel_circ_0003944, novel_circ_0001458 and novel_circ_0000707), and based on them, potential circRNA-miRNA binding pairs were predicted. Sanger sequencing, RNase R treatment experiment and qPCR validation identified the accuracy of the circRNAs. Key circRNAs identified in the present study were covalently closed and were more stable under RNase R treatment than linear RNAs. Based on function analysis, circRNAs could regulate metabolic process, signal transduction, and ion responses in A. japonicus when exposed to thermal and hypoxic stress, and ‘regulation of response to stimulus’ is a common gene ontology (GO) term that is significantly enriched in each treatment; GO terms for ‘DNA’ and ‘stress’ are commonly enriched in heat-related treatments (HT and HL); and GO terms for ‘protein’ are commonly enriched in hypoxia-related treatments (LO and HL). When environmentally stressed, ‘metabolism,’ ‘transport and catabolism,’ ‘membrane transport,’ and ‘signal transduction’ were significantly responded in sea cucumber based on KEGG analysis. We provide insights into circRNA functions in stress regulation and lay a foundation for invertebrate circRNA research.

CircRNAs play an important regulatory role in the regulation of disease. However, we have a limited understanding of the role of circRNAs in the host's complex protective and pathological mechanisms of BVDV infection. Transcriptome analysis of circRNAs in host cells after BVDV infection may allow us to understand the biological functions of circRNAs in the regulation of BVDV infection. Here, we identified a total of 19,118 circRNAs from the MBDK cells (at 12 h, 24 h, and 48 h post-infection) infected with BVDV by using RNA-seq technology. We confirmed several circRNAs using RT-PCR and DNA sequencing, and qRT-PCR analysis was performed to identify several circRNAs expression and circRNAs resistance to RNase R digestion. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that the host genes of differentially expressed circRNAs were involved in the regulation of cell proliferation, apoptosis, cycle and viral infection related signaling pathways. These results indicate that circRNA in host cells plays a broad regulatory role after BVDV infection and provides a valuable resource for studying circRNA biology in host cells after BVDV infection.

Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3′-5′ exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3′ ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases.

Bacterial and eukaryotic hibernation factors prevent translation by physically blocking the decoding center of ribosomes, a phenomenon called ribosome hibernation that often occurs in response to nutrient deprivation. The human pathogen Staphylococcus aureus lacking the sole hibernation factor HPF undergoes massive ribosome degradation via an unknown pathway. Using genetic and biochemical approaches, we find that inactivating the 3′-to-5′ exonuclease RNase R suppresses ribosome degradation in the Δhpf mutant. In vitro cell-free degradation assays confirm that 30S and 70S ribosomes isolated from the Δhpf mutant are extremely susceptible to RNase R, in stark contrast to nucleolytic resistance of the HPF-bound 70S and 100S complexes isolated from the wild type. In the absence of HPF, specific S. aureus 16S rRNA helices are sensitive to nucleolytic cleavage. These RNase hot spots are distinct from that found in the Escherichia coli ribosomes. S. aureus RNase R is associated with ribosomes, but unlike the E. coli counterpart, it is not regulated by general stressors and acetylation. The results not only highlight key differences between the evolutionarily conserved RNase R homologs but also provide direct evidence that HPF preserves ribosome integrity beyond its role in translational avoidance, thereby poising the hibernating ribosomes for rapid resumption of translation. IMPORTANCE Ribosome hibernation is pivotal for the rapid recovery of translation after quiescence in both bacteria and eukaryotes. Ribosome hibernation factors sterically occlude the entry of mRNA and tRNA and are thought to primarily maintain ribosomes in a translation-repressive state, thereby providing a pool of readily recyclable 70S or 80S complexes upon dissociation of the hibernation factors. Ribosomes in Staphylococcus aureus cells lacking the sole hibernation factor HPF are extremely unstable. Here, we show that HPF binding inhibits ribosome degradation by the evolutionarily conserved exoribonuclease RNase R. The data not only uncover a direct protective role of HPF in ribosome stability but also reinforce the versatility of RNase R in RNA processing, decay, and ribosome quality control.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs which are mainly formed by reverse splicing of precursor mRNAs. They are relatively stable and resistant to RNase R because of their covalently closed structure without 5’ caps or 3’ poly-adenylated tails. CircRNAs are widely expressed in eukaryotic cells and show tissue, timing, and disease specificity. Recent studies have found that circRNAs play an important role in many diseases. In particular, they affect the proliferation, invasion and prognosis of cancer by regulating gene expression. CircRNA Forkhead box O3 (circFOXO3) is a circRNA confirmed to be abnormally expressed in a variety of cancers, including prostate cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, and breast cancer, etc. At present, the feature of circFOXO3 as a molecular sponge is widely studied to promote or inhibit the development of cancers. However, the diverse functions of circFOXO3 have not been fully understood. Hence, it is important to review the roles of circFOXO3 in cancers. This review has summarized and discussed the roles and molecular mechanism of circFOXO3 and its target genes in these cancers, which can help to enrich our understanding to the functions of circRNAs and carry out subsequent researches on circFOXO3.

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete thus permitting the re-initiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

Objective: Aberrantly expressed exosomal circular RNAs (circRNAs) have been reported in various human cancers. Nevertheless, it remains elusive in cutaneous squamous cell carcinoma (cSCC). Herein, based on RNA-seq, we systematically uncovered the expression and implication of exosomal circRNAs in cSCC.Methods: Plasma exosomes derived from cSCC and healthy subjects were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot. Differentially expressed exosomal circular RNAs (circRNAs) were screened by RNA-seq analysis, which were validated by RT-qPCR. Among them, the biological structure of circ-CYP24A1 was validated by Sanger sequencing and RNase R digestion. Si-circ-CYP24A1 was transfected into exosomes, followed by incubation with A431 and SCL-1 cells. Then, viability, apoptosis, migration, and invasion were evaluated by CCK-8, TUNEL staining and migration assays.Results: This study identified 25 up- and 76 down-regulated exosomal circRNAs in cSCC than healthy subjects. Among them, circulating circ-CYP24A1 was confirmed to be up-regulated in cSCC. Circ-CYP24A1 had a covalently closed circular structure and was not sensitive to RNase R digestion. After incubation with si-circ-CYP24A1-transfected exosomes, proliferation, migration, and invasion were lowered while apoptosis was enhanced in A431 and SCL-1 cells. Meanwhile, si-circ-CYP24A1-transfected exosomes significantly decreased the expression of downstream targets CDS2, MAVS, and SOGA in cSCC cells.Conclusion: Collectively, our study identified that targeting exosomal circ-CYP24A1 could suppress cSCC progression by weakening tumor malignant behaviors, which might provide a promising therapeutic target and non-invasive diagnostic biomarker for cSCC.