Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA–DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity.Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In  In vitro, it also catalyses the reverse template-independent NDP polymerization reaction leading to de novo synthesis of RNA molecules or 3′-end tailing of pre-existing RNAs3. Escherichia coli PNPase (hereafter EcPNPase) in vitro activity on RNA and its cellular function in RNA turnover and in the control of complex phenotypic traits, like biofilm formation and adaptation to the growth at sub-optimal temperature, have been extensively studied by different groups over the many years since bacterial PNPase discovery5. Global mRNA profiling of E. coli mutants lacking EcPNPase by RNA-Seq showed that the enzyme controls—directly or indirectly—the level of around 200 transcripts, with the 59% of RNAs increased and the others decreased in EcPNPase absence6. In a previous genome-wide analysis of E. coli transcription profile performed by microarray, an even higher number of RNAs whose level was modulated by EcPNPase was found (i.e. more than 500)7. Auto-regulation of the EcPNPase pnp gene is considered a paradigmatic example of post-transcriptional regulation. EcPNPase promotes pnp mRNA degradation by digesting a short non-coding RNA that protects it from RNase E-dependent endonucleolytic cleavage8. EcPNPase regulates its own expression also via other pathways not requiring the enzyme phosphorolytic activity, namely by binding the pnp mRNA and eliciting premature transcription termination and probably, translational repression12. Far less understood aspects related to EcPNPase are how (and if) small molecules reported to bind and modulate EcPNPase in vitro impact the enzyme activity in the bacterial cells16, and the EcPNPase role in DNA repair and oxidative stress response. In vitro, the enzyme degrades ssDNA molecules in presence of Pi and Mn2+ producing dNDPs. It can also catalyse template-independent polymerization of dNDPs and 3′-end tailing of DNA molecules18. In vivo, EcPNPase enhances homologous recombination, and pnp mutants are sensitive to UV radiation and defective in DNA repair of double-stranded breaks20. The mechanistic function of EcPNPase in DNA repair is unknown. It is still unclear also its role in the protection against oxidative stress, which was imputed to EcPNPase-dependent removal of oxidized RNA, for which such enzyme has high affinity21.Polyribonucleotide phosphorylase (PNPase) is an evolutionarily conserved phosphorolytic exonuclease that degrades the RNA from 3′- to 5′-end in presence of phosphate (Pi) and Mg23. hPNPase is essential in mice and human cells, and severe pathological conditions with a wide spectrum of symptoms are caused by mutations in the hPNPase PNPT1 gene27. RNA import from cytoplasm into mitochondria and degradation of mitochondrial RNAs are the main activities credited to hPNPase fractions located in the intermembrane space and in the matrix, respectively28. Interestingly, also EcPNPase can switch from an RNA degrading to an RNA carrier (and stabilizing) mode depending on the RNA substrate and the interaction with other proteins like the DEAD-box RNA helicase RhlB, which can unfold RNA secondary structures inhibiting EcPNPase activity30. Similarly, in the mitochondrial matrix hPNPase forms a complex with the DExH-box RNA helicase SUV3 able to efficiently degrade dsRNA33. The complex is involved in mitochondrial DNA homeostasis, a function that has been linked to its ability of removing R-loops, i.e. stable RNA–DNA hybrids displacing a DNA strand and potentially interfering with DNA replication34. Whether the human enzyme can bind and degrade DNA like its bacterial counterpart is unknown. Conversely, it is known that hPNPase can bind, but not degrade, oxidized RNA36. hPNPase is 40% identical to EcPNPase . Indeed, hPNPase phosphorolytic activity was reported to be inhibited in vitro at phosphate concentrations in the range of those found in the E. coli cytoplasm40.The studies on the human orthologous protein (hereafter hPNPase) began much more recently, when the human PNPase (hPNPase) cDNA was cloned and the protein shown to localize in mitochondria38, we analysed the phenotype and global transcription profile of an E. coli strain expressing the human enzyme instead of the natural orthologue. We found that hPNPase cannot complement, and actually worsens, several phenotypes due to the lack of EcPNPase. hPNPase deeply impacts E. coli gene expression and triggers the SOS response, most likely by eliciting oxidative stress and accumulation of R-loops.In this work, to get insight into hPNPase activity in a cellular context simpler than that of the human cell and containing high Pi41 an E. coli strain expressing human PNPase instead of the natural EcPNPase from the pnp chromosomal locus  from the pnp promoter produced an mRNA with the same 5′-UTR as the pnp transcript and the hPNPase open reading frame (ORF) with the E. coli codon usage15.We constructed by λ RED recombinationPNPT1Ec cultures had longer generation time (g) in exponential phase and reached lower optical density after 24 h than both pnp+ and Δpnp cultures  markers, compatible with the predicted MW of hPNPase lacking the mitochondrial localization signal 45. We did not investigate the mechanism determining low abundance of the tagged hPNPase, which could be due to decreased mRNA or protein stability.To compare the level of hPNPase in the sed Fig. C. Based pnp null mutations in E. coli. In particular, we assayed (i) aggregation of cultures growing in minimal medium with glucose as sole carbon source, detectable as a drop in the optical density of late exponential cultures46. This phenotype was tested also at low Pi concentration, which should increase and decrease, respectively, hPNPase and EcPNPase phosphorolytic activity28; (ii) grow inhibition at temperatures below 18–20°C47; (iii) increased sensitivity to H2O2, causing oxidative damage, and to mutagenic agents like UV rays and zeocin, an antibiotic inducing DNA double-strand breaks (DSBs)21; and iv) reduced frequency of spontaneous mutations20. As shown in Fig. PNPT1Ec cultures growing in minimal medium started decreasing when the cultures were at OD600 = 0.2–0.3, whereas the Δpnp strain aggregated at higher cell density. Interestingly, we observed that low Pi concentration promoted the aggregation of the pnp+ strain, whereas it prevented PNPT1Ec aggregation and had a modest effect, if any, on the Δpnp strain  with FDR < 0.01 and |log2(FoldChange)|≥ 1 as thresholds  < − 3) were genes belonging to tna and tdc operons controlling tryptophan and threonine degradation, and the glpABC genes encoding the anaerobic glycerol-3-phosphate dehydrogenase49. Most of the genes with highest relative expression in PNPT1Ec (log2(FoldChange) > 3), like tisB, umuD, sulA, dinI, recN and recA, belonged to the SOS response, a regulatory mechanism that allows the coordinated induction of tens of genes, mainly involved in DNA repair, in presence of DNA damage50.To assess the impact of hPNPase on lds Fig. . DEGs foPNPT1Ec and the Δpnp strains and maintaining similar expression in presence or absence of the EcPNPase (i.e. in the pnp+ vs. Δpnp comparison)  Fig. B. These 1Ec Fig. B,C. LexAOverall, these data indicate that hPNPase has differential activity with respect to the bacterial enzyme and that may cause genotoxic and membrane stress.50. Constitutive induction of the SOS response in PNPT1Ec indicates that this strain may have high levels of ssDNA, which typically accumulates when cells attempt to replicate damaged DNA.The SOS response is induced by LexA repressor self-cleavage stimulated by activated RecA (RecA*), which is a RecA filament assembled on a ssDNA scaffoldE. coli by Reactive oxygen species (ROS), which are the major source of endogenous DNA damage54. Considering that hPNPase induces ROS when overexpressed in human cells55, we speculated that hPNPase may either induce ROS or interfere with ROS detoxification. In fact, ROS levels measured using the fluorescent probe DCFH-DA resulted significantly higher in PNPT1Ec than in pnp+  of the PNPT1Ec, pnp+ and Δpnp strains immediately before (T0) and 4 min after blocking transcription initiation with rifampicin (T4). The residual RNA amounts of the different E. coli RNA species at T4 should depend on the stability of each of them in the three strains. Stability was thus estimated for each gene according to the percentage of its RNA found remaining at T4, expressed as the percentage of the CPM values at T0 maintained at T4 . Conversely, the transcripts more stable in the presence of hPNPase were more than double those stabilized in the presence of EcPNPase . These data are consistent with a very limited, if any, RNA degrading activity of hPNPase in E. coli cells.As shown in Fig. E. coli strain that carries the PNPT1Ec gene encoding the human PNPase inserted into the pnp locus. Our data clearly show that human PNPase cannot compensate the phenotypic effects due to the lack of the natural EcPNPase. On the contrary, the PNPase of spinach chloroplast complemented the cold sensitive phenotype of an E. coli pnp mutant62. This difference may be due to higher similarity of chloroplast PNPase to EcPNPase, as both the RPH2 and the KH-S1 domains of EcPNPase are more similar to those of spinach PNPase than to hPNPase ones. Indeed, only spinach PNPase, and not hPNPase, can form heterotrimers with EcPNPase subunits62. Since residues 327–331 of EcPNPase, with which RNase E forms hydrogen-bonding interactions63, are not conserved in the human enzyme, it is very unlikely that hPNPase could be recruited in the RNase E-based RNA degradosome. Thus, it seems sound that hPNPase cannot replace the endogenous enzyme in all those activities in which EcPNPase participates as a component of the multisubunit RNA degradosome.In this work, we characterized an pnp and the PNPT1Ec strains showed that the bacterial enzyme caused the destabilization of more than 500 transcripts, whereas only 27 were modestly destabilized in PNPT1Ec, suggesting that the human enzyme may be substantially devoid of degradative activity in the bacterial context. Previous in vitro analyses showed that the phosphorolytic activity of hPNPase is strongly impaired when Pi concentration is greater than 2.5 mM37. Since in the cytoplasm of E. coli growing cells Pi concentration is around 10 mM38, this would explain why hPNPase has no degradation activity in the bacterial cell. Consistent with this hypothesis, at low Pi, which should improve hPNPase phosphorolytic activity and worsen that of EcPNPase, the strong aggregative phenotype shown in M9 by PNPT1Ec is suppressed, whereas the pnp+ strain aggregates. Although these data should be taken with some caution, giving the pleiotropic effects that Pi concentration modulation may have, they support the hypothesis that hPNPase may be inactive in E. coli because of high Pi level. Interestingly, Pi should be at about 10 mM in the mammalian cell cytoplasm65 and, as a consequence, also in the mitochondrial intermembrane space, as this compartment has similar composition to the cytosol with respect to ions and small molecules to which the mitochondrial outer membrane is highly permeable67. Indeed, phosphate concentration in whole mitochondria has been estimated to be greater than 2.5 mM69, implying that hPNPase phosphorolytic activity should be essentially inhibited or limited to sub-mitochondrial regions in which Pi concentration is locally and/or temporarily low. In agreement with this hypothesis, mitochondrial RNA (mtRNA) degradation by the hPNPase-hSUV3 complex, namely the mitochondrial degradosome, has been reported to take place in relatively few spots inside the mitochondrial matrix32.The comparison of total RNA stability between the ΔPNPT1Ec strain does not simply recapitulate the phenotype of strains with loss of function mutations in the pnp gene. Phenotypic traits due to EcPNPase lack, like the fitness decrease, the aggregation in minimal medium, the sensitivity to agents causing—directly or indirectly—DNA damage like UV, zeocin, and H2O2, the low frequency of spontaneous mutations and the ROS accumulation are all exacerbated by hPNPase, and more than 200 genes are differentially expressed between the PNPT1Ec and the Δpnp strains. These findings are consistent with hPNPase retaining some activity in E. coli cells, and we speculate that it may retain RNA binding activity, which is not necessarily linked to catalysis28. Binding by hPNPase could interfere with RNA function and RNA degradation pathways active in the absence of EcPNPase worsening the effect of the Δpnp mutation. A limitation of this work is that we have not assayed the effect of a PNPT1Ec mutation eliminating the RNA binding domains of hPNPase. This experiment, which we plan to do, will allow to test our hypothesis that at least some PNPT1Ec phenotypic traits may be due to ectopic RNA binding.Lack of catalytic activity and impaired interaction with other components of the bacterial RNA degradation machinery explain why hPNPase cannot compensate for the lack of EcPNPase. However, the PNPT1Ec slow growth play a major role in RNA stabilization and only a fraction of the RNAs responsive to hPNPase are actually direct targets of the enzyme, the effect of hPNPase on overall RNA stability remains striking, with many RNAs (i.e. about the 42%) more stable in presence of hPNPase in PNPT1Ec than in Δpnp mutant. Paradoxical RNA stabilizing effect exerted by E. coli exonucleases, among which EcPNPase, was already reported70 and it has been confirmed by our data, as around 700 RNAs (i.e. about the 19%) were more stable in the presence of EcPNPase in the pnp+ than in its absence in the Δpnp strain.Even assuming that indirect effects linked to the 55. Our data suggest that hPNPase may exert the same effect in E. coli, making unlikely that hPNPase-dependent ROS induction may require the interaction with specific human proteins or RNAs.Intriguingly, overexpressed hPNPase promotes ROS in Hela cells through an unknown mechanismPNPT1Ec strain, the SOS response is constitutively active. Since the SOS response is elicited by RecA*71, i.e. a filament of RecA protein assembled on ssDNA, this indicates that hPNPase stimulates ssDNA accumulation. A relevant source of ssDNA in the PNPT1Ec are most likely the R-loops that are abundant in such strain. Accordingly, some phenotypic traits of the PNPT1Ec, and in particular SOS induction and slower growth in aerobiosis, are also shown by rnhA mutants lacking RNase HI57. In principle, hPNPase may indirectly affect R-loop level by down-regulating the expression of the RNase HI rnhA gene or of recG and topA genes encoding RecG and topoisomerase I, respectively, which have also been implicated in R-loop homeostasis72. However, transcriptomic data show that rnhA, recG and topA genes are all equally expressed in presence or absence of hPNPase, ruling out their down-regulation by hPNPase, at least at the level of mRNA synthesis and/or degradation. Moreover, rnhA mutants do not grow in anaerobiosis57, whereas the PNPT1Ec does, suggesting that the presence of hPNPase does not abolish RNase HI activity. hPNPase may indirectly promote R-loops because it causes ROS accumulation, which is known to induce R-loops in human cells75. However, R-loops are abundant also in anaerobiosis, suggesting that other mechanisms can contribute to their formation. Factors binding the nascent RNA co-transcriptionally, like Rho, have been shown to play a pivotal role in avoiding excessive R-loop formation78. hPNPase ectopic binding of RNAs emerging from transcriptional bubbles may interfere with the anti-R-loop activity of such factors. On the contrary, in human cells, hPNPase loading onto the nascent transcript would prevent the formation of R-loops or destabilize them because hPNPase would bring to the transcript the SUV3 helicase, which is able to denature RNA–DNA hybrids79.In the PNPT1Ec and pnp long mRNAs in the PNPT1Ec and pnp+ strains, respectively, hPNPase was less abundant than EcPNPase. EcPNPase negatively regulates its own expression through complex post-transcriptional mechanisms modulating pnp mRNA translation and stability, which mainly occur at the level of the mRNA 5′-UTR1. Thus, while it is not surprising that replacing EcPNPase with hPNPase impacts autoregulation, given the dissimilar activity of the two proteins, we would have expected mRNA stabilization and increased translation in presence of hPNPase, as shown by strains expressing mutated EcPNPases82. It is possible that despite codon optimization for E. coli, translation elongation of the PNPT1Ec mRNA is not efficient and impairs hPNPase expression even though autoregulation is not stringent. The abundance of the short PNPT1Ec RNA in the PNPT1Ec  unless differently stated. C-6001 was obtained by replacing in C-1a42 the pnp ORF (region 3309033–3311168) with a PNPT1Ec:kanR cassette by λRED recombination41. PNPT1Ec:kanR was constructed by assembling amplicons deriving from PCR amplification with ad hoc primers of PNPT1Ec and kanR cassette on pET28b-H6-hPNPase and pKD13, respectively41. PNPT1Ec encodes hPNPase ORF with the E. coli codon usage and without the first 45 amino acids corresponding to the mitochondrial localization signal. All other strains were similarly constructed by assembling in vitro, by PCR amplification, recombinant cassettes and inserting them into the E. coli chromosome by λRED recombination. FLP recombination was exploited to remove antibiotic resistance genes in all strains41. The PNPases expressed by C-6009 and C-6011 have 6 residues long His-tag at the C-terminus. For pETD-hPNP construction, PNPT1Ec was amplified by PCR on pET28b-H6-hPNPase with proper primers and cloned in pETDuet-1 digested with NcoI-EcoRI, by NEBuilder Hi-Fi DNA Assembly (New England Biolabs).85 or M9/2.5 i.e. M9 with a ca. 40-fold reduction of the phosphate content (from 57 to 1.3 mM) and 0.4% glucose. When needed, media were supplemented with 100 µg/ml ampicillin, 50 µg/ml kanamycin, 0.1 mM IPTG. Growth at low oxygen in liquid cultures was performed by inoculating bacteria in 15 ml conical Falcon tubes filled to the brim with LD medium with screwed caps and further sealed with parafilm. Incubation was performed static at 37 °C. In this condition, given low solubility of oxygen in culture medium and poor diffusion, bacteria below about 1 mm grow anaerobically86. Growth in anaerobiosis in solid media was performed by incubating the plates at 37 °C in an airtight jar in presence of an Aerogen (Oxoid) sachet.Bacterial cultures were grown in LD broth, M9 supp, i.e. M9 minimal medium with 0.4% (w/v) glucose and 2.5% LD87. For hPNPase stability determination, overnight cultures were diluted to OD600 = 0.02 in LD broth and grown at 37 °C, aerated. At OD600 = 0.6, 25 µg/ml streptomycin was added, and 2 ml samples were collected every 20 min for 1 h. Samples were pelleted and resuspended in protein loading sample buffer  to OD600 = 25. After 3 min boiling, protein samples  were loaded onto 10 or 12% SDS–polyacrylamide gel, as stated in Figure legends. After the run, the gels were blotted onto PROTRAN nitrocellulose membranes  and the membranes stained with Ponceau S solution (Sigma). Immunodecoration was performed with one of the following antibodies diluted in Blotto87 as indicated in figure legends: (i) monoclonal anti-hPNPase (Anti-hPNP: PNPase (G-11) sc-365049; Santa Cruz Biotechnology); (ii) polyclonal anti-hPNPase ; (iii) polyclonal anti-L4 or S3 ribosomal proteins ; (iv) polyclonal anti-EcPNPase88 (v) anti-His tag antibody .Proteins were extracted by sonication from mid-exponential cultures grown in LD at 37 °C as described32P]-ATP and T4 polynucleotide kinase were previously described90. Oligonucleotide probes used for Northern blotting were CPXQ (4416706–4416727); RECA (1059766–1059783); SOXS (4245855–4245873); 1842 (5S-specific primer)91. RT-PCR (Reverse Transcription- PCR) was performed on RNA extracted as for the RNA-Seq. 2 μg were reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen). 1:40 cDNA dilution was used for PCR with primers specific for either the PNPT1Ec mRNA or 16S rRNA (used as reference gene87), respectively. RT-qPCR (Reverse Transcription- quantitative PCR) was performed on RNA extracted from three independent cultures for each strain. 1 μg of RNA was reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen) and 1:10 cDNA dilution was used for RT-qPCR with SYBR Premix Ex Taq (Takara) with primers specific for the PNPT1Ec mRNA. Two technical duplicates were performed for each biological replicate and reactions were run in a CFX Connect Real-Time PCR Detection System (Bio-Rad). 16S rRNA was used as reference gene to normalize the results and calculate the relative fold change in gene expression using the CFXmaestro Software (Bio-Rad).Procedures for RNA extraction, electrophoresis on denaturing polyacrylamide and agarose gels, Northern blotting, staining of the RNA on filters with methylene blue and 5′-end labelling of oligonucleotides with [γ92 from three replicate cultures of C-1a, C-5691 and C-6001 grown in LD at 37 °C up to OD600 = 0.8. Each RNA sample was qualitatively and quantitatively checked on Agilent 2100 Bioanalyzer RNA Pico Chip . Directional RNA-Seq libraries were prepared from 1 µg of total RNA using the TruSeq Stranded Total RNA with Illumina Ribo-Zero Plus Sample Prep Kit  according to the manufacturer’s protocol. Sequencing was performed on an Illumina NextSeq 500 platform  with 12 M 2 × 75 paired end reads for each sample. For RNA stability analysis, three replicate cultures of C-1a, C-5691 and C-6001 were grown in LD at 37 °C up to OD600 = 0.8 and rifampicin (0.4 mg/ml) was added to the cultures. Samples for RNA extraction were taken immediately before (time 0) and 4 min after the addition of the antibiotic. The RNA was processed for library preparation and sequencing on Illumina NextSeq 500 platform as described before. For all datasets, sequencing reads were mapped on the E. coli K12 MG1655 RefSeq genome assembly (NCBI accession number NC_000913.3) with bowtie293 in “very sensitive” mode allowing a maximum insert size of 1000 bps. Expression quantification was performed with respect to the latest gene annotation retrieved from NCBI (GCF_000005845.2_ASM584v2). Transcript levels of each gene were defined as the number of reads overlapping its transcribed region, computed with bedtools94. Differential expression analysis was performed with edgeR95. Initial read counts were normalized by trimmed mean of M values (TMM), with default parameters. Differentially expressed genes were identified by the quasi-likelihood (QL) F-test of edgeR . We selected as differentially expressed all genes with an adjusted p-value (FDR) < 0.01 and a log fold change higher than 1 or lower than -1 . For RNA stability analysis, raw reads counts were normalized by library size and counts per million employed in subsequent comparisons.Global transcript levels were determined by extracting total RNA as described600 = 0.8) grown in LD at 37 °C were harvested, resuspended in 200 µl of PBS containing 1.0 mM DCFH-DA (Sigma-Aldrich) and incubated 30 min at 37 °C. The samples were then washed threefold with PBS and transferred into a 96-well plate. The OD600 and fluorescence at 488/530 (excitation/emission) nm were measured with an EnSight (PerkinElmer) plate reader. Fluorescence was normalized by the OD600 of the tested culture sample.200 µl of exponential cultures (OD78. 10 μg samples were immobilized on Hybond-N+ nylon membrane (PerkinElmer) using a Dot-blot apparatus (Hybri-Dot Manifold). RNase treatment was performed before immobilization on the membrane by incubating the samples with 20 U of Ribonuclease H (NEB) 3 h at 37 °C or 1U of RNase III (Epicentre) for 1 h at 37 °C. In this condition, RNase III visibly degrades 8 µg of total RNA as determined in preliminary experiments (not shown). After UV crosslinking, the filters were immunodecorated with the RNA–DNA hybrid specific S9.6 antibody (EDM Millipore) diluted 1:5000 in blocking buffer (5% milk in TBST). Incubation was performed either 3 h at room temperature or overnight at 4 °C with similar results. After washing, incubation with the secondary anti-mouse IgG antibody (Thermo Fisher Scientific) was performed 1 h at room temperature. Signal detection was carried out via chemiluminescence (PDS standard ECL GeneSpin) using ChemiDoc Touch Imaging System (Bio-Rad) for membrane imaging. After the acquisition, the membrane was washed in water, soaked in 5% acetic acid for 15 min with shaking, and the nucleic acids stained with 0.05% methylene blue dissolved in 0.5 M sodium acetate buffer (pH 5.2).Total nucleic acids extraction from 10 ml overnight cultures grown in LD at 37 °C and R-loop detection were performed essentially as described96 from the median of the titer obtained in LD plate (N) and the median of the number of mutated colonies obtained on streptomycin plates (r), by resolving the equation: A fluctuation test was performed to evaluate streptomycin resistance mutation rate . Independent cultures of C-1a, C-5691 and C-6001 (N = 12) were incubated overnight at 37 °C and titers were measured by plating proper dilutions on LD-Agar plates. The undiluted cultures were also plated on LD-Agar plates containing 0.025 mg/ml streptomycin. Mutations per culture value (m) was calculated as describedP < 0.05.The replicate number for each sample are indicated in the figure legends, when appropriate. Statistical significance was determined by two-tailed Student's t-test when comparing two groups or ANOVA with post-hoc Tuckey test when comparing three or more groups. Pearson’s Chi-square test was used to compare distributions. The differences in means were considered statistically significant at Supplementary Figures.Supplementary Table S1.Supplementary Table S2.


Elucidation of the cellular functions of RNAs represents one of the most exciting research areas in modern life sciences. RNAs play crucial roles in plant biology, including developmental regulation and environmental responses. Advances in sequencing technologies have benefitted the genome-wide analysis of RNA modifications, the resolution of RNA secondary structures, the mapping of epigenetic modifications, the identification of RNA-edited sequences, and the discovery of novel RNA classes. These advances have allowed impressive progress in our understanding of cellular roles of RNAs, but have also given rise to new questions . In this special issue, we have collected expert contributions that provide updates on different aspects of plant RNA biology, thereby highlighting the most intriguing open questions and the future direction of this exciting field of research.RNA polymerases synthesize multiple distinct RNA species in cells. RNA is synthesized from either DNA templates by DNA-dependent RNA polymerases (RDPs) or RNA templates through RNA-dependent RNA polymerases (RDRs). Five nuclear RDP polymerase (NRP) complexes (NRPA–E) generate different types of RNAs from genomic DNA with functional roles across the cell (RDRs generate double-stranded RNAs (dsRNAs) from single-stranded RNA (ssRNA) templates. Cellular infection strategies of plant pathogens may rely on intermediates. dsRNAs are a potent trigger for cellular pathogen defence pathways of plant cells, namely RNA interference. The Arabidopsis genome contains six RDRs linked to the silencing of distinct target RNAs . RDR actR-loops represent DNA–RNA structures where RNA invades the DNA double-strand . HoweverSplicing offers a key mechanism to shape the expression of distinct messenger RNA (mRNA) isoforms from the DNA sequences of single eukaryotic genes , perhapsDNA sequences in plant genomes that elicit transcription termination and 3´-end formation represent ’terminators’ . Similar to splicing, alternative termination and alternative poly-adenylation (APA) of mRNA generate different isoforms of mRNAs  uses a fluorescent probe to label single RNA molecules in cells, and the technique thus offers information on the cellular RNA localization and expression levels. In this special issue, Transcription of genes by Pol II into mRNA provides the substrate for ribosomal translation into proteins. Ribosomes are composed of protein and ncRNA subunits critical for translation. Cellular pathways control the journey of mRNAs from the nucleus to the cytoplasmic ribosomes, and the ribosomal activity. These mechanisms of cellular regulation offer an explanation for the differences that can exist between protein levels in cells estimated based on mRNA expression. Ribosomes engaged in translation are purified from cells in polysome fractions, and the association of mRNAs in polysomes thus offers insights into their translation. Here, The multiple cellular roles of RNAs offer tantalizing avenues for biotechnological exploitation. Included in this special issue is a study that highlights the advantages and opportunities of RNA biotechnology in the context of agriculture , ribosomal RNAs (rRNAs), long non-coding RNAs (lncRNAs), and small RNAs (sRNAs) . The stulncRNAs are a class of ncRNAs comprising molecules with a length greater than ~200 nt that do not encode for stable proteins , small nucleolar RNA (snoRNAs), small nuclear RNAs (snRNAs), and the recently discovered tiny RNAs (tiRNAs)  proteins to form RNA-induced silencing complexes (RISCs) to fulfill their regulatory actions. How a miRNA or siRNA will act largely depends on which AGO protein loads them. All plants encode multiple copies of AGO proteins with different preferences for the length, the 5’-nucleotide, and the subcellular distribution of the sRNA. Each of these AGOs will later act differently depending on its nature and the loaded sRNA . For exaInterestingly, many ncRNAs, especially the sRNAs, can exit one cell and act autonomously in another. This movement and cross-cellular action are particularly relevant during development to create the gene expression gradients required for the proper formation of tissues and organs. Such movement is also crucial for the systemic defense against pathogens, especially viruses. But the most impressive recent discovery has been that of the capacity of certain organisms to interact with each other, both parasitically and symbiotically, using extracellular RNA molecules (exRNAs) (This special issue highlights many of the multiple roles RNA species play in plants. The world of RNAs in plant cells is complex. While many are needed for gene expression and protein production, they are also critical for cellular resilience, and yet they also provide a platform for successful infection strategies by pathogens. Strikingly, after decades of RNA research, the purpose of most ncRNAs produced in cells remains to be determined. RNAs are moving into public perception, but they also literally move within cells between and across the whole plant. Entire pathways are dedicated to regulating the cellular activities of RNAs, indicating that there are clearly advantages for cells in relying on RNA-based regulation. Elucidating the molecular principles underlying this RNA-based regulation in plants and the benefits RNA-based solutions offer to plant cells for solving environmental challenges during growth promises to inspire biotechnological solutions for current barriers in agriculture.


PLOS Biology almost 20 years ago, established a framework to understand how multiple RNA interference pathways can regulate the genome in parallel.Cells from most eukaryotic species make several different types of small interfering RNAs. Pioneering work in plants, published in  This Perspective looks back at a classic PLOS Biology paper that provided an early glimpse of the complexity of RNAi pathways in plants and how it acted as a springboard for further exploration of this extravagant universe. PLOS Biology 20th Anniversary Collection.This article is part of the It’s a cliché repeated at the beginning of many papers that plants “can’t run.” Plant researchers, on the other hand, move very fast indeed, often leaving animal biologists far behind as they push molecular biology into exciting new territory. Remarkably, despite the extraordinary evolutionary distance and the entirely separate evolution of multicellularity in the two groups, many of the discoveries made in plants later turn out to be conceptually similar or even homologous in animals.RNA interference (or RNAi) is a striking example where plants “got there first,” uncovering a transformative molecular mechanism that turned out to be essential for animals too. The basic process of RNAi involves a small noncoding RNA, which, in conjunction with a member of a family of proteins known as Argonautes, regulates a target mRNA, leading to its silencing . The souThe central process of RNAi was first discovered in plants and characterised as a form of antiviral defence. The double-strand RNA could come directly from RNA viruses; single-strand RNA viruses will produce double-strand RNAs as part of their replication cycle . Later, PLOS Biology in 2004, to clearly demonstrate that these paralogues really were at the heart of diversity in RNAi [One of the key properties that underpins all this flamboyance is the existence of several parallel mechanisms to process and partition the many pathways of RNAi. The most conceptually straightforward way in which this can occur is through paralogues with dedicated specificity. Early on, it was recognised that plants contain several Dicer and RNA-dependent RNA polymerase paralogues; however, it took a major advance, published in  in RNAi . Xie and in RNAi . A simplIn addition to showing distinct genetic requirements for these pathways, the paper also made some important observations that turned out to be prescient in understanding RNAi pathway diversity across eukaryotes. In 2004, the high-throughput sequencing methods that are so ubiquitous now were not readily available. Nevertheless, using standard cloning and sequencing techniques, Xie and colleagues assayed over 1,000 different endogenous small noncoding RNAs corresponding to the DCL3 pathway. They showed that these RNAs had a modal length of 24 nucleotides, contrasting to the modal length of 22 nucleotides that characterises antiviral small noncoding RNAs .C. elegans, the one dicer enzyme carries out antiviral RNAi, endogenous RNAi, and microRNA biogenesis, with cofactors and different downstream Argonaute proteins delimiting the pathways [Many gaps remained to be filled in. We now know that RDR1, RDR2, and RDR6 all contribute to viral defence to some extent . A fourtpathways . Indeed,pathways . NeverthPLOS Biology paper to unravel!From a personal perspective, my interest in the evolutionary aspects of small noncoding RNA pathways can trace its descent to the pioneering experiments of Xie and colleagues. While they were studying RNAi in one species, the existence of multiple pathways coexisting often in the same cell emphasises the evolutionary plasticity of RNAi. The specialisation of the paralogues implies that a common ancestral RNAi pathway, perhaps involving one Dicer, one RNA-dependent RNA polymerase, and one Argonaute, can evolve rapidly, with new pathways such as the transposon-controlling piRNAs emerging in animals , and new


Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca2+-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure.Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3′UTRs as well as c-myc 5′UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the KD of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3′UTR with KDs in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5′UTR indicating some selectivity.Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca2+. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context. Annexins (Anxs) are multi-functional and -compartmental proteins participating in a variety of cellular functions related to cell proliferation, membrane-cytoskeleton interactions, endo- and exocytosis, the formation of exosomes, as well as mRNA transport and translation . Membran2+-dependent manner  and paraspeckle component 1 (PSPC1) to paraspecles in the nucleus  together with proteins and regulatory RNAs . The proThe mRNAs are transported in large complexes often consisting of a set of mRNAs participating in the same cellular process . Some prhttp://www.exocarta.org/)  in small vesicles known as exosomes, which represents a mode of epigenetic regulation of gene expression. Exosomes are a special type of secretory membrane vesicles , releaseta.org/) . AnxA2 ita.org/)  and exosta.org/) . AnxA2 ata.org/) . Regulatta.org/)  stronglyta.org/) . At presIn addition to proteins and specific mRNAs, exosomes may transfer soluble factors and miRNAs  and evenin vitro transcribed regions of specific mRNAs. The obtained results are supported by the determination of the KDs of selected Anxs.Since our initial experiments analyzing the interaction between AnxA2 and mRNA showed that the RNA-binding site is located in domain IV of the conserved structural core, we speculated that the RNA-binding property could be a common feature of several Anxs, if not shared by all of them. To investigate this possibility, we cloned all rat Anxs and investigated their ability to bind mRNA directly, both to oligo (dT) affinity-purified total mRNA from PC12 cells and NcoI and Acc65I , purified after separation in a 1% agarose gel and ligated into the same restriction sites of the pETM10 vector. All restriction enzymes used were FastDigest Restriction Enzymes . N-terminally truncated versions of AnxA7 (Δ142AnxA7) and AnxA11 (Δ188AnxA11)   were genE. coli BL21 (DE3) bacteria were transformed with pETM10 plasmids containing the respective anx cDNAs. The bacteria were grown in 300 mL LB medium with 50 μg/mL kanamycin until they reached an OD600 of 0.6. Subsequently, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce protein expression. The N-terminally truncated versions of AnxA7 and AnxA11 were both induced at 25°C for 4 h, while wt Anxs were expressed ON at 15°C. Following incubation, the bacteria were collected by centrifugation at 4,000 g for 15 min. The pellets were frozen at −80°C and thawed in Breakage buffer  glycerol, 1 mM DTT) before the addition of 0.5 μg/mL DNase I, 0.25 μg/mL RNase A and EDTA-free protease inhibitor cocktail . Subsequently, the bacteria were sonicated on ice 8 × 30 s and the lysates were centrifuged at 16,000 g for 30 min at 4°C. All purification steps were performed at 4°C. The supernatants from the cleared bacterial lysates were loaded onto Ni2+-NTA or Co2+-NTA  agarose columns and incubated for 30 min on a turning wheel. Purification on Co2+2+ instead of Ni2+ resin resulted in less impurities in the AnxA7, AnxA9 and AnxA11 protein samples. Purification of the Anxs was performed essentially as described  containing 1 mg/mL yeast tRNA and 0.4 U/µL RNasin  or Ribolock RNase inhibitor . Subsequently, the fraction was incubated for 60 min with oligo (dT) magnetic beads  at 4°C on a turning wheel. The beads were then washed three times with tRNA-free RNA-binding buffer. Polyadenylated mRNAs with bound proteins were eluted from the beads by incubation for 10 min at 65°C in 60 µL elution buffer (pre-heated at 65°C) containing 0.1% SDS and 1 mM DTT. The elution step was repeated once, and the mRNAs in the two combined fractions were degraded by incubation with 0.3 μg/μL RNase A for 10 min at 30°C to facilitate the separation of proteins by SDS-PAGE for immunoblot analyses.The cytoskeletal fraction (∼800 μg protein) of PC12 cells was diluted 1:4 in RNA-binding buffer (10 mM triethanolamine (pH 7.4), 50 mM KCl, 1 mM DTT, 2 mM MgSO32P] inorganic phosphate  per T75 flask. The medium was poured off and the cells were resuspended in 9 mL Trizol reagent. In the next step, the suspension was transferred to a Corex tube, incubated for 5 min at RT and 2 mL chloroform was added per mL Trizol. The sample was shaken vigorously by hand and incubated for 3 min at RT before centrifugation at 12,000 g for 15 min at 4°C. The aqueous phase was transferred into a new Corex tube, and the radiolabeled RNA was precipitated by using 0.5 mL isopropanol per mL Trizol and incubated for 10 min at RT. The precipitated RNA was collected by centrifugation at 12,000 g for 10 min at 4°C. The supernatant on top of the RNA pellet was discarded. Subsequently, the RNA pellet was washed in cold 70% (v/v) EtOH and centrifuged at 7,500 g for 5 min at 4°C. After drying, the RNA pellet was dissolved in 30 μL ddH20 and incubated for 10 min at 70°C. The sample with total RNA was divided in smaller aliquots and stored at −80°C. Total mRNA was affinity-purified from total RNA using Dynabeads (dT)25  according to the procedure given by the manufacturer.Confluent PC12 cells (5 x T75 flasks) were incubated for 60 min in a phosphate-free medium  supplemented with dialyzed 15% fetal calf serum and 5 mCi -rUTP  was incorporated during transcription to label the transcripts. The RNA transcripts were subjected to agarose gel electrophoresis to confirm the transcription of correctly sized RNAs and to ensure that the RNAs were not degraded. For details, see (8 cpm/μg transcript was achieved.) vector . The cDNeviously . The conils, see . Typical32P] metabolically labeled total RNA or total mRNA derived from rat PC12 cells or in vitro transcribed [32P] rUTP-labeled transcripts of anxA2 mRNA (full-length), c-myc 3′UTR or the CDS of hRLuc mRNA. Protein samples (3 µL) were spotted onto nitrocellulose membranes (0.2 µm pore size) in three different concentrations  and air-dried. The samples of AnxA2 that were denatured, were heat-inactivated at 95°C for 5 min. The membranes were incubated overnight in 1xDenhard’s solution  in RNA-binding buffer  containing tRNA (1 mg/mL) to block unspecific binding of RNA. The RNA transcripts  were preheated for 3 min at 72°C, and then gradually cooled for 15 min to RT to allow folding of secondary structure and subsequently incubated together with (1 u/mL) RiboLock RNase Inhibitor  with the proteins on the spot blot for 30 min at RT on a tilting disk. The RNA-binding buffer containing radiolabeled RNA was removed and the membranes were quickly rinsed three times with RNA-binding buffer without tRNA. The membranes were further washed 4 × 15 min in the same buffer. The membranes were air-dried for 30 min and exposed to phosphor-imager screens . Bound RNA was detected by scanning in a Phosphor-Imager .The RNA-binding abilities of the Anx proteins were studied by exposing nitrocellulose membranes spotted with the twelve rat Anx proteins in their native form to [in vitro transcribed [32P] rUTP-labeled transcripts of anxA2 or c-myc 3′UTRs) was assayed by UV-crosslinking experiments using the RNA-binding buffer described in The interaction of the Anxs with the RNAs  and incubated at 37°C for 2 h. 0.2 M NaBH4 and 1 M Tris-HCl, pH 8.2 were added and the reaction was incubated for 30 min on ice in the dark. The RNA was precipitated as described above and resuspended in ddH2O. The RNA samples were immobilized onto High Precision Streptavidin (SAX) biosensors  by dipping into 40 nM RNA solution for 2 min at a stir rate of 1,000 rpm. The reference sensors were prepared by dipping SAX biosensors into 10 µM of biocytin solution. The proteins of interest were prepared in RNA-binding buffer at a dilution series of 0, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 µM. Binding was monitored on the OctetRED96 instrument  by allowing the association for 180 s and the dissociation for 200 s. The data were analyzed for a 1:1 binding model using fortéBIO Octet data analysis software (version 9). Briefly, the response of each RNA immobilized sensor for protein analytes was subtracted with the response for the same sample from the biocytin immobilized reference sensors. This was followed by baseline correction and subsequently applying Savitzky-Golay filtering according to the application note “Biomolecular Binding Kinetics Assays on the Octet® BLI Platform by Sartorius. The kinetic local fitting was applied to an individual group of unique sensor locations using a 1:1 model. The fitness of the data to the model was judged by the reduced χ2 and R2 values and KD values were reported for R2 values >0.9.The anxA2 and c-myc 3′UTRs were biotinylated at the 3′-end using periodate-chemistry. In brief, the 2′,3′ diols of the escribed . 5 μM RNSDS-PAGE was performed using 10% or 4%–15% gradient gels  and the denaturation buffer used was from BioRad (Cat. 1610747) added 100 µL β-mercapto ethanol per 900 µL buffer. The composition of the denaturation buffer is 277.8 mM Tris-HCl (pH 6.8), 44.4% (v/v) glycerol, 4.4% LDS, 0.02% bromophenol blue. The proteins were transferred onto nitrocellulose membranes (0.2 µm pore size) by blotting performed using the Trans-Blot Turbo Transfer System  essentially according to the manufacturer . The membranes were probed against AnxA1 , AnxA2 , AnxA4 , AnxA5 , AnxA6 , AnxA7 , AnxA10 , AnxA11 , AnxA13 ), Rab7 , Rab11 , EEA1 , S6 , and PABP1  primary antibodies. Rabbit polyclonal antibodies against SPC25 was a generous gift from Stephen High . Proteins were detected by incubation at a 1:2,000 dilution with secondary horseradish peroxidase (HRP)-conjugated antibodies  or 170–6,515 (goat anti-rabbit) from Bio-Rad, Hercules, United States) or from Santa Cruz, Dallas, United States . The reactive protein bands were visualized using the WesternBright ECL HRP substrate .Our previous studies revealed that AnxA2 is enriched in the cytoskeletal fraction. Furthermore, we showed that a subpopulation of AnxA2 in this fraction is associated with mRNAs in polysomes and monosomes  and partThus, we first isolated polysomes from the cytoskeletal fraction  derived D of about 250 nM when assayed alone. Its presence in mRNP complexes derived from PC12 cells shows that it is associated with RNA  magnetic beads . HoweverNext, we assayed the binding of all Anxs to full-length rat anxA2 mRNA , which wSince in this experimental set-up all Anxs are spotted on the same membrane and therefore compete for the same RNA, their relative different affinities for RNA are also revealed to a certain degree. This depends, however, on the proportion of degraded and/or denatured protein in each spot since all steps before drying of the membranes and exposure to screens include several overnight incubations. It is also possible that those Anxs, which bind RNA with low affinity will not be readily revealed due to the lack of UV-crosslinking and thorough washing.Finally, the binding of the Anxs to the coding region of Renilla luciferase mRNA was additionally investigated as a negative control  since prin vitro UV-crosslinking method employing purified recombinant Anxs and purified in vitro transcribed (32P) rUTP-labeled RNA . This leads to an extremely slow off rate . AnxA2, Δ142AnxA7 and Δ188AnxA11 were chosen as Anxs showing a known or an overall strong binding to RNA, while AnxA4 was chosen for its apparent low or lack of binding to RNA. AnxA13 was selected as an Anx with an apparent relatively intermediate binding to RNA  mRNA . In PC12This was most evident for AnxA2 and AnxA13 . AnxA1, What is the function of the Anxs in mRNP complexes? Of interest, regarding AnxA2, it has been reported to associate with poly(A) binding protein 1 (PABP1) . Further2+-bound dimer of AnxA2 are exposed at the side of the structure that is opposite to the site of dimerization (pdb: 1XJL)  .UV-crosslinking of RNA to the Anxs does not lead to detectable protein retardation during 4%–15% SDS-PAGE, indicating the crosslinking of a maximum of 4–6 RNase-resistant nucleotides . Using tIn addition to UV-crosslinking experiments, we also used binding assays involving spotting of the native proteins in three concentrations onto the same membrane (immobilized phase) for incubation with the radio-labeled transcript in solution . Thus, tDs of selected Anxs were determined using the real-time BLI. Biotinylated c-myc 5′UTR, c-myc or anxA2 3′UTRs were immobilized onto the biosensor surface while the Anxs  were kept in solution. All the selected Anxs , except AnxA4, interacted with the c-myc and anxA2 3′UTRs with KDs in the nM range. Regarding the interaction between Δ188AnxA11 and anxA2 3′UTR the KD was found to lie in the µM range .Thus, while a single RNA-binding site has been revealed in AnxA2 , it is pAn attractive functional implication of our present findings is that Anxs play a role in the coordinated transport of vesicles and mRNAs to specific subcellular locations to co-regulate cellular responses to specific signals. Thus, multiple Anxs may tether the mRNAs to vesicles as first shown for AnxA11 . Recent In conclusion, - using different experimental approaches we show - that the direct RNA-binding property–first discovered and characterized in the case of AnxA2 —appears


Hepatocellular carcinoma (HCC) is difficult to distinguish from hepatocellular adenoma (HCA) in dogs, and HCC may develop from HCA, according to recent reports. Therefore, urgent research is needed to establish a reliable biomarker for the early detection of these hepatic diseases. Noncoding RNAs (ncRNAs) could be a very useful tool for diagnosing hepatic diseases. Y RNA is a regulatory RNA type with a sequence of 80–110 nucleotides. In this study, we investigated novel Y RNA-derived fragments, namely Y RNA, which we previously investigated in canine mammary gland tumors and found that they could differentiate between benign and malignant tumors. Accordingly, we decided to investigate Y RNA in canine HCC and HCA, which has not been attempted before in either humans or dogs. We report that Y RNA can discriminate canine HCC from HCA and could be a promising biomarker for diagnosing canine HCC and HCA.Hepatocellular carcinomas (HCC) are common tumors, whereas hepatocellular adenomas (HCA) are rare, benign tumors in dogs. The aberrant expression of noncoding RNAs (ncRNAs) plays a pivotal role in HCC tumorigenesis and progression. Among ncRNAs, micro RNAs have been widely researched in human HCC, but much less widely in canine HCC. However, Y RNA-derived fragments have yet to be investigated in canine HCC and HCA. This study targeted canine HCC and HCA patients. We used qRT-PCR to determine Y RNA expression in clinical tissues, plasma, and plasma extracellular vesicles, and two HCC cell lines (95-1044 and AZACH). Y RNA was significantly decreased in tissue, plasma, and plasma extracellular vesicles for canine HCC versus canine HCA and healthy controls. Y RNA was decreased in 95-1044 and AZACH cells versus normal liver tissue and in AZACH versus 95-1044 cells. In plasma samples, Y RNA levels were decreased in HCC versus HCA and Healthy controls and increased in HCA versus Healthy controls. Receiver operating characteristic analysis showed that Y RNA could be a promising biomarker for distinguishing HCC from HCA and healthy controls. Overall, the dysregulated expression of Y RNA can distinguish canine HCC from HCA. However, further research is necessary to elucidate the underlying Y RNA-related molecular mechanisms in hepatocellular neoplastic diseases. To the best of our knowledge, this is the first report on the relative expression of Y RNA in canine HCC and HCA. Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can occur in both canine and human patients . HCAs arNoncoding RNAs (ncRNAs) are potentially implicated in hepatocellular tumorigenesis and may serve as a diagnostic marker for these tumors ,9. ncRNAY RNA is first reported in patients with systemic lupus erythematosus in 1981 . DespiteThe dysregulation of Y RNAs may contribute to the development of tumors, affect cell growth, and promote inflammation . Y RNAs Y RNAs have been found in substantial amounts in plasma and serum from human patients ,29, otheExtracellular vehicles (EVs) are of potential interest for the quantification of Y RNA fragments and other ncRNAs. They are small structures released by cells to facilitate the transportation of vital components, such as DNA, RNA, and proteins, for effective intercellular communication . EV-deriTGF-beta, seem to be evident in the development of HCC in dogs and humans [Similar gene expression patterns, such as the significance of d humans . That isAccordingly, in this study, we aimed to determine relative Y RNA expression levels in dogs with HCC and HCA using qRT-PCR analysis, targeting tumor tissues, plasma, and plasma EVs from clinical samples, and HCC cell lines, to evaluate Y RNAs as diagnostic biomarkers for these two types of liver tumor in dogs. n = 15) or HCC (n = 13) by a veterinary pathologist when undergoing surgery at the Kagoshima University Veterinary Teaching Hospital or an affiliated clinic, between September 2012 and December 2022. The owner of each dog gave informed consent the use of samples in this research. Samples were also collected from nine healthy adult laboratory beagle dogs to include as healthy controls in the evaluation provided by Shin Nippon Biomedical Laboratories, Ltd.  [The clinical samples evaluated in this study had been obtained from a population of 28 dogs (age range: 8–14 years) diagnosed histo-pathologically with HCA . Full details of the HCA and HCC patients are summarized in g for 10 min to remove the cell debris. The plasma samples were separated and centrifuged again at 16,000× g at 4 °C to remove the debris. The supernatant was transferred to new Eppendorf tubes and stored at −80 °C as plasma samples.Tumor tissue samples were collected at the time of surgery from the clinical patients, and biopsy samples were collected from the livers of healthy controls. Plasma samples were obtained from a subset of the study population  and 0.25% trypsin or 0.1% EDTA were applied for detachment of the cells. Cells were counted using an automated cell counter . In this study, we evaluated two HCC cell lines, 95-1044 (a fast-proliferating cell line) and AZACH (an intermediate-proliferating cell line) ,44. Cellg for 5 min. The supernatant was discarded, and the tube was centrifuged again at 1000× g for 30 s to remove the residual reagent. Finally, the pellet was reconstituted in 150 μL 1X PBS and stored at −80 °C for further analysis.The Total Exosome RNA and Protein Isolation Kit  was used to isolate EVs from plasma, following the manufacturer’s protocol. In brief, 300 μL plasma samples were mixed with a half volume of 1X PBS, 90 μL of exosome precipitation reagent was then added, and the resultant mixture was vortexed thoroughly and centrifuged at 10,000× TM RNA Isolation Kit  was used to extract total RNA from tissues and cells in accordance with the manufacturer’s protocol. A mirVana PARIS Kit (Thermo Fisher Scientific) was used to isolate total RNAs from plasma samples and EVs. Before extraction, 5 μL (5 femtomoles) of synthetic cel-miR-39 was added to every plasma and plasma EV sample for normalization. Briefly, each tissue sample or the relevant HCC cell preparation was mixed with the required amount of lysis buffer. A 300 µL aliquot of each plasma sample was mixed with an equivalent amount of 2× denaturation solution. A 1:10 ratio of a miRNA homogenate additive was added to the tissue or cell lysate, then kept on ice for 10 min. An amount of 600 µL Acid-phenol:chloroform (Ambion®) was added to the tissue, cell lysate, or plasma, with subsequent thorough vortex-mixing and then centrifugation at 15,000× g for 5 min at room temperature. The supernatant was then collected carefully in an Eppendorf tube, to which a 1.25-fold amount of molecular-grade ethanol (99.9% in purity) was added (and the amount recorded), and the tube contents were filtered using centrifugation. In the final step, total RNA was obtained as sediment in the tube using an elution solution pre-heated to 95 °C. The NanoDrop 2000c spectrophotometer was used to measure the concentration of total RNA (Thermo Fisher Scientific). To evaluate the quality and integrity of RNA, an Agilent 2100 Bioanalyzer was utilized . The cells and tissues had RNA Integrity Numbers ranging from 8.5 to 9.5.A mirVana−ΔΔCT method was used. RNU6B was used as an internal control for tissues and plasma, miR-16 was for the plasma, and miR-186 was for EVs [Y RNA was selected based on a previously published NGS dataset (SRA: PRJNA716131) for canine mammary gland tumors . The qRT for EVs . The Taqhttps://www.graphpad.com/, accessed on 1 August 2023) was used for statistical analysis and graph visualization. A Mann–Whitney U test and a one-way analysis of variance (ANOVA) followed by the Kruskal-Wallis test were used to assess the qRT-PCR results where applicable. ROC curves and AUCs were plotted using Wilson/Brown method. A p-value < 0.05 was considered statistically significant.GraphPad Prism 9 (p = 0.008) versus healthy controls (p = 0.001) versus HCA. However, the Y RNA expression level did not significantly differ between healthy controls and HCA. Thus, the expression profile for Y RNA in HCC differed to those in healthy controls and HCA.The relative expression of Y RNA was investigated in HCA and HCC tissue samples. YRNA was significantly decreased in HCC (fold change (FC) = 0.43, controls . In addip = 0.002) and significantly increased in HCA  versus Healthy controls (p = 0.030) versus HCA. Taken together, our findings indicate that Y RNA expression may differentiate HCC and HCA from Healthy controls and HCC from HCA.We evaluated the expression of the selected Y RNA in plasma samples. Y RNA was significantly decreased in HCC  versus Healthy controls , and HCA   . Howeverp = 0.0002) and AZACH  cells versus normal liver tissue (p = 0.004) versus AZACH cells. Our results thus suggest that Y RNA expression is substantially decreased in fast-proliferative HCC cell lines, which is consistent with the results for clinical tumor tissue samples.The relative expression of Y RNA was evaluated in fast-proliferative 95-1044 and intermediate-proliferating AZACH cell lines. Y RNA was significantly decreased in 95-1044  and 1.00 (p = 0.004) for HCA and HCC, respectively, when evaluated against Healthy controls (p = 0.004) from HCA in plasma samples (p = 0.003) from Healthy controls  curves and areas under the curve (AUCs) were generated to investigate the diagnostic value of Y RNA. In plasma analyses, Y RNA yielded AUCs of 0.920  from benign tumors (HCA) and tumor-free patients. However, this study still has some limitations. First, our study sample was relatively small. We need to validate Y RNA in a large cohort sample to strengthen our findings further. Second, the roles of Y RNA in canine HCC development need to be investigated in a bio-functional study.Pet dogs have great potential utility for comparative oncology clinical trials, partly because they maintain an intact immune system and experience natural co-evolution of the tumor microenvironment . Humans To our knowledge, this is the first report on Y RNA in canine HCC and HCA. This ncRNA has distinctive characteristics and differentiates malignant tumors (HCC) from benign tumors (HCA). Notably, its expression pattern is consistent across clinical samples and cell lines. We thus consider that Y RNA has promising potential for differentiating HCC from HCA. Our findings provide significant insights into how Y RNA contributes to the progression of hepatic disease in dogs. Further research is required to fully elucidate the role of Y RNA in the development and progression of canine HCC and HCA.


Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.Anaerobic, mesophilic, and cellulolytic cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3′ end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon.In this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.Our findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in The online version contains supplementary material available at 10.1186/s13068-023-02357-5. Ruminiclostridium cellulolyticum (previously Clostridium cellulolyticum), a cellulosome-producing and anaerobic cellulolytic Gram-positive bacterium, is one research model organism and a promising host for biofuel production from lignocellulose [cip-cel operon in R. cellulolyticum harbors 12 genes that encode the major cellulosomal subunits that are essential for cellulose degradation. Transcriptional analysis of the cip-cel operon showed several transcripts displaying different stabilities were produced suggesting the processing of cip-cel mRNA [cip-cel mRNA was processed initially into several segments, and then, variation in the stability between these segments contributed to their differential expression [ellulose –4. The cellulose –7. The ccel mRNA . Our datpression .rpsU-dnaG-rpoD [glmU-glmS [LEE4 [Escherichia coli, and cagA [Helicobacter pylori. In the gram-positive Bacillus subtilis, the bicistronic mRNA of the glycolysis operon cggR-gapA was shown to be cleaved near the end of cggR resulting in an unstable cggR portion and a stable gapA portion [cdd-bmpA [speB [Streptococcus pyogenes and saePQRS [Staphylococcus aureus. As a general rule, RNA processing is initiated via primary cleavage by an endoribonuclease, leading to the generation of decay intermediates, and variation in stability in the segments generated by exoribonuclease contributes to differential gene expression.In bacteria, numerous mRNAs are processed to mature forms like rRNAs and tRNAs, and this is a key event regulating these genes at the post-transcriptional level, often in response to environmental constraints. During the process of RNA maturation, RNases and small RNAs (sRNAs) that would otherwise promote RNA degradation convert labile RNAs into stable and biologically functional molecules . ComparenaG-rpoD , 12, glmlmU-glmS , and LEEmS [LEE4  in Escheand cagA  in Helic portion , 17. Simcdd-bmpA , and spepA [speB  in Strep saePQRS  in StaphE. coli and related Gram-negative species, initiating RNA processing is mainly accomplished by the endoribonuclease RNase E, which is essential for cell viability [B. subtilis and S. aureus) where RNase E is absent, RNase Y has been proposed to be the enzyme responsible for the cleavage of bulk mRNAs and to be the functional equivalent of RNase E [Streptococcus pyogenes and Staphylococcus aureus prefers to cleave downstream of a G based on RNA-seq data. And RNase Y was found to cleave in single-stranded regions close to secondary structures, such as the saePQRS operon in S. aureus [E. coli RNase E was shown to almost completely restore wild-type growth in an rny mutant of B. subtilis [B. subtilis has been shown to initiate decay of the small trp leader RNA by cleaving the upstream of the 3′ transcription terminator [thrS mRNA, causing an increase in downstream thrS mRNA stability [In iability , 22 and iability , 23–25.  RNase E . It has . aureus , 20, 27.subtilis , suggestrminator , 29 and tability –34. Althcip-cel transcripts. Here, we show that RNA cleavage events happen at six intergenic regions harboring stem-loop structures in the cip-cel operon and the secondary structures at both ends of processed transcripts contribute to their stability. Cloning analysis revealed that all cleavage of cip-cel mRNAs was determined by their nearby stem-loops, which included two distinct types of stem sequences. Thus, our study shows that the processing of cip-cel transcripts is controlled by stem-loop structures that can be recognized by endoribonucleases and determine the location of the cleavage sites.The aim of this work was to elucidate the sequence/structural requirements of processing for cip-cel operon spanning 26 kb in the genome of R. cellulolyticum harbors 12 genes that encode the major cellulosomal subunits that are essential for cellulose degradation. Gene cipC, encoding a cellulosomal scaffolding, is the first gene of this operon and is followed by 11 other genes , most of which encode cellulases  between cipC and cel48F, cel48F and cel8C, cel9G and cel9E, and cel9E and orfX, resulting in secondary transcripts having different abundances due to their difference in stability  and mcherry (encoding a red fluorescence protein)  detected by both probes, suggesting that they were not processed  and upstream of IR4 (named IR4::SL2), resulting in processed mcherry from IR2 with SL2 at its 5′ end or processed fbfp from IR4 with SL2 at its 3′ end. We found processed monocistronic transcripts of mcherry from IR2 and fbfp from IR4 that could not be detected by Northern blotting, but were clearly detected after the introduction of SL2  upstream of the reporter gene  upstreamcip-cel mRNA processing, we constructed three IR1 derivatives by, respectively, deleting their two stem-loops and the linker between them . These constructs were also inserted into our fbfp and mcherry reporter system, and processing was analyzed by Northern blotting , the RNA cleavage was completely abolished , which were potentially key sequences for RNA cleavage Fig. a. To verhed Fig. b, c. Howrry Fig. d, e. Thumcherry was significantly decreased when both 2-GC-pair regions, especially GC2, were deleted, suggesting that both 2-GC-pair regions of IR5-SL1 were important for RNA cleavage, similar to IR1-SL1. However, the abundance of upstream monocistronic transcripts of fbfp was hardly changed when the 2-GC-pair regions were deleted  in addition to two 2-GC-pair regions at the top and bottom. It was showed that the deletion of the GC3 (named IR5-SL1∆GC3) did not prevent cleavage of IR5, but when it was deleted together with GC2 (named IR5-SL1∆GC2∆GC3), the abundance of monocistronic transcripts of fbfp was significantly decreased, suggesting that GC3 is important to transcription termination . The results of Northern blotting indicated that the replacement of the GC1 or GC2 region led to a significant reduction in the transcript level of monocistronic bfp Fig. i. It is To confirm the role of these two 2-GC-pair regions, they were respectively added to the top and bottom of a stem-loop (SLx) that is supposed not be cleaved, and the addition of 2-UA-pair regions were used as two control Fig. j. As expfbfp and mcherry in derivatives of IR2 were much lower than that of wild-type IR2 transcripts  was also predicted upstream of the cleavage site in IR4, in addition to the large stem-loop (named SL2) involved in RNA cleavage Fig. a.Fig. 6Mfbfp that were hardly detected in IR4 were detected decisively, and the abundance of monocistronic transcripts of mcherry was significantly decreased when SL1 in IR4 was deleted (IR4ΔSL1)  Fig. b, which L1) Fig. c. The reion Fig. d, in whiion Fig. e, the RNion Fig. f, g. Thufbfp was increased, while that of mcherry was decreased as the AT-pair region was abolished , while only processed monocistronic transcripts of fbfp were detected when the intact SL2 was replaced (IR4ΔSL2::IR1-SL1) . The results from Northern blotting showed that the RNA cleavage efficiency of these two derivatives was decreased dramatically compared to IR4  and Ccel_RS08950 (rnj), respectively. However, the gene rny encoding RNase Y could not be disrupted by clostron [R. cellulolyticum, which was consistent with findings in other Gram-positive bacteria such as B. subtilis and Clostridium perfringens [rnj encoding RNase J can be disrupted by clostron but this does not affect the cleavage of cip-cel mRNA  [R. cellulolyticum, similar to other Gram-positive bacteria, such as B. subtilis, in addition 3′-to-5′ exoribonucleases . In contrast, no processive RNA degradation activity in the 5′-to-3′ direction has been demonstrated in E. coli [cip-cel mRNA was further confirmed by the rnj mutant, in which the transcript level of the cip-cel operon was much higher than wild-type, suggesting that RNase J contributes to the stability of the cip-cel mRNA  medium. R. cellulolyticum ATCC35319 (H10) was cultured anaerobically at 35 °C in GS-2 medium supplemented with 3 g L−1 of cellobiose as the sole carbon source [600 of 0.8–1.0. When required, antibiotics were added at the following concentrations: 100 μg mL−1 ampicillin or 20 μg mL−1 erythromycin.The bacterial strains and plasmids used in this study are listed in Additional file n source . The culmcherry gene (encoding a red fluorescence protein) by overlapping extension (SOE) PCR using synthetic oligonucleotide primers with R. cellulolyticum genomic DNA used as a template. The fused fragments were subsequently digested with BglII and EcoRI and cloned between the corresponding site of pMCT6 [fbfp and mcherry and they were expressed in a single operon, driven by the Pthl promoter (the thiolase gene promoter from Clostridium acetobutylicum) [R. cellulolyticum cells were electro-transformed anaerobically with all plasmids according to the literature protocol [−1 erythromycin. All primers used are listed in Additional file All plasmids constructed in this study are listed in Additional file of pMCT6 . In the tylicum) . R. cellprotocol  and tranR. cellulolyticum gene Ccel_RS08950 (encoding nuclease RNase J) was constructed from clostron plasmid pSY6 [http://clostron.com/) [Ccel_RS08950.pSY6-RS08950 for targeted disruption of mid pSY6 . The taron.com/) . The cloR. cellulolyticum cultures on cellobiose using an EZ-10 Total RNA Mini-Prep Kit . Five micrograms of RNA samples were separated via electrophoresis on 1% denaturing gel agarose gel  and blotted onto a N+ nylon membrane  using the Northern MaxTM-Gly kit Complete Northern Blotting . Cy5.5-labelled DNA probes for the detection of fbfp and mcherry were directly prepared commercially  using the oligonucleotides in which Cy5.5 was coupled to the 5′ end of the oligonucleotides as listed in Additional file Total RNA was isolated from mcherry, a sequencing ladder starting at the 5′end of the transcript was run in parallel. In the case of the full-length transcript, the second fragment originating from this cleavage site has a predicted length about 150 bp corresponding to the sequences between the cleavage site and the 3′end of the transcript.The processing site was mapped by reverse transcription of the processed transcript using an oligonucleotide  and the Cy5.5-labeled oligonucleotide primer were denatured 5 min at 65 °C and subsequently incubated on ice for 2 min. Samples were reverse transcribed using 200 U of Hifair IV reverse transcriptase  in the presence of 5× first strand buffer, 5 mM dNTP Mix and 40 U of Recombinant Ribonuclease Inhibitor , as per the manufacturer’s instructions. Thermal cycling conditions were as follows: 25 °C for 5 min, 55 °C for 15 min and 85 °C for 5 min. The cDNA products were analyzed on an 8% polyacrylamide sequencing gel (8 M urea/TBE). Sequencing ladders were generated using USB Thermo Sequenase Cycle Sequencing Kit  with the same Cy5.5-labeled oligonucleotide primer, and the recombinant plasmids digested by BglII is as template.fbfp and mcherry was measured via qRT-PCR. The cDNA product was obtained by qRT-PCR using the Hiscript ® III RT SuperMix for qRT-PCR (+gDNA wiper) kit as described by the manufacturer’s instructions. The qRT-PCR was performed using ChamQ Univeral SYBR qPCR Master Mix  on CFX96 real-time PCR detection system  and the results normalized via abundance of Ccel_RS01560 encoding the β subunit of DNA-directed RNA polymerase. The primer sets for qRT-PCR are listed in Additional file To analyze the effect of the introduction of IRs and their derivatives to transcription of the upstream and downstream genes in the dual-fluorescence reporter system, the relative transcript level of −1 of rifampicin  was added to exponentially growing R. cellulolytiucm cells to prevent transcription initiation. Samples for RNA preparation were removed before and at different times  after rifampin addition, and preparation of total RNA was carried out as described previously [fbfp or mcherry expression for each mutation at the given time points after rifampin addition.For mRNA stability assays, 80 μg mLR. cellulolyticum in NCBI (ID: NC_011898). The full RNA secondary structure of the sequences as defined above was computed using Mfold with default parameters . The output secondary structures were collected and analyzed for the presence of stems and loops.The sequences of the intergenic regions (IRs) are exactly from the first nucleotide after stop codon of the upstream gene to the last nucleotide before start codon of the downstream gene according to the genome annotation of Additional file 1: Figure S1. Secondary structure prediction of all intergenic regions (IRs) from cip-cel operon. Figure S2. RNA cleavage sites were precisely identified by primer extension assay. Figure S3. Analysis of the stability of fbfp mRNA. Figure S4. The effect of RNase J on RNA cleavage of the cip-cel operon. Table S1. Strains and plasmids used in this study. Table S2. Primers used in this study. Table S3. Sequences of IRs in the cip-cel operon.


Hepatitis E virus (HEV) genotype 7 is a zoonotic disease detected in dromedary camels.The consumption of camel meat and dairy products, the abundance of dromedary camels in Southeast Iran and the import of camels from neighbouring countries to Iran made the researchers investigate the infection rate of camels by the virus.A total of 53 healthy camels in Southeast Iran  tested for HEV RNA.A total of 17 blood samples and 36 liver samples were taken from 53 healthy dromedary camels (aged between 2 and 10 years) from various southeastern regions of Iran. The samples were tested for HEV using RT‐PCR.n = 30) tested positive for HEV RNA.Overall, 56.6% of the studied samples  from dromedary camels in Southeast Iran using reverse transcription polymerase chain reaction, which might play the role of a zoonosis reservoir for its transmission to humans. The southeastern region of Iran  was selected for the study as half of dromedary camels in Iran and camels imported from Pakistan and Afghanistan are in this region. The samples were taken randomly from camels in Zabol, Saravan and Mirjaveh Counties, Sistan and Baluchistan province with the largest camel population. Sampling was carried out over multiple periods during January–June 2019. The blood samples were taken from the jugular vein of apparently healthy dromedary camels after clinical examinations and with the consideration of the ethical principles, and liver samples were taken from apparently healthy camels after slaughter. Each sample's information, including age, gender and geographical location, was collected and recorded using specific questionnaires.Seventeen blood samples and 36 liver samples were obtained from apparently healthy dromedary camels (aged between 2 and 10 years) in Southeast Iran. About 58.5% of the camels were male, and 41.5% of them were female. The DENAzist column RNA isolation kit  was used according to the manufacturer's instructions to extract RNA from the liver samples, and the DENAzist whole blood RNA isolation kit was used to extract RNA from the blood samples. Following the RNA extraction, 5 μL of the extracted RNA along with 1 μL of loading buffer‐dye 6X were loaded onto 1.5% agarose gel to ensure the quality and non‐degradation of RNA bands. A NanoDrop device was used for the quantitative study of the extracted RNA. After confirming the RNA extracted from camel liver and blood, the RNA purification was carried out using the DNase  commercial kit according to a kit protocol to eliminate possible DNA contaminations. The purified RNAs were then used for cDNA synthesis using the Pars Toos commercial kit. All stages, from RNA extraction to cDNA synthesis, were carried out immediately after sampling on the same day. The extracted samples were stored in freezers at −80°C for the subsequent examinations. The Actin Beta‐like2 gene (ACTBL2) was used as a reference gene to ensure high‐quality cDNA synthesis. The gene was found to be 100% present in dromedary camels after blast analysis. After alignment and thermodynamic evaluation of the primers, the forward and reverse gene reference primers 5′‐TATTGGCAACGAGCGGTTCC‐3′ and 5′‐GGCATAGAGGTCTTTACGGATGTC‐3′, respectively, were selected. An ordinary PCR was performed with the reference gene primers and synthesized cDNAs at 54°C to verify cDNA quality. After loading the result on agarose gel, the presence of bands in the range of the length of reference gene primers’ fragment (139 bp) indicated the quality of the synthesized cDNAs Figure . The PCR3n = 30) tested positive for HEV RNA using the RT‐PCR method. Statistical data analysis using the Fisher exact test indicated a significant relationship between the camels’ hepatitis E infection and their geographical location (p < 0.001) so that the infection rate was higher in camels from Mirjaveh County than in those from other counties. The results also indicated the high frequency of infection in camels imported from Pakistan and Afghanistan. The camels’ infection with hepatitis E also revealed a significant relationship with their age (p < 0.001) so that the highest infection rate was observed in camels younger than 2 years old. The infection rate in the liver tissue was 65.71% (23/35) as opposed to the 38.89% (7/18) infection in the blood; however, the infection rate showed no significant relationship with the type of sample (p = 0.058). The frequency of HEV was revealed to be 67.74% (21/31) in male dromedary camels as opposed to 40.90% (9/22) in female dromedary camels; this difference was statistically significant (p = 0.048)  tested positive for HEV RNA. Previous reports indicated that the DcHEV prevalence was 1.5% (3 out of 203 stool samples) in the Middle East using the RT‐PCR method in 2014 . The tests indicated a higher frequency of infection in camels from border areas and camels imported from Pakistan and Afghanistan. Rasche et al.  between HEV infection in male and female camels, with a higher number of infected males. El‐Kafrawy et al. (p = 0.015); hence, male camels were more likely to be infected than female camels (31.6% vs. 13.4%) . According to Corman et al., Our study found a significant relationship between the distribution of camels’ hepatitis E infection and their age group so that the infection rate was significantly higher in camels under 2 years of age compared to other ages .https://publons.com/publon/10.1002/vms3.1174.The peer review history for this article is available at


For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two—SETD1A and SETD1B—contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs. Histone modifications are finely tuned molecular mechanisms by which many basic nuclear processes are controlled. These epigenetic marks are placed on specific residues in the disordered histone tails by designated enzymes and collectively create an intricate regulatory pattern responsible for the implementation of complete genetic programs. One of the most prevalent histone modifications is lysine methylation, which is carried out by histone lysine methyltransferases (HKMTs), a large group of enzymes with more than fifty members in the human genome, each responsible for the placement of a specific histone lysine mark. This modification in turn induces the recruitment of transcription factors or directly influences chromatin structure, thus achieving alterations in gene expression. Proteins with histone lysine methylation activity all contain a SET domain—with the exception of DOT1L—which is responsible for the catalytic activity ). Statistical analysis was performed using GraphPad Prism 8.0.1, one-way ANOVA.Experiments were carried out on at least three independent biological replicates and the fold enrichment of immunoprecipitated RNAs was calculated as 2p values were calculated by unpaired two-tailed Student’s t-test, and p < 0.05 to p < 0.0001 were marked by * to ****, respectively.All experiments were carried out on at least three independent biological replicates and distinct samples were selected for relative measurements. For quantification of colocalization intensity, all images were taken randomly from the same sample, and relative quantifications were reproduced from independent biological replicates to recover similar results. The nuclear speckle protein condensates or cytoplasmic SETD1A clusters were gated as regions of interest (ROIs) for the measurement of Pearson’s coefficient. Using GraphPad Prism 8 , statistical Our presented results show for the first time that SETD1A is capable of interacting with several RNA molecules in the cells. We reproductively showed that the isolated RRM domain is capable of RNA binding on its own, in contrast with the yeast Set1 RRM. We also provided structural analysis of the possible RNA binding surfaces on the SETD1A RRM domain and identified the main differences between these regions in SETD1A and Set1, explaining the inability of the latter to interact with RNAs on its own.Nevertheless, our studies also have some limitations that need to be taken into consideration and investigated further. One such shortcoming is the lack of information on the essentiality of the RRM domain for RNA binding within the protein. It is becoming more widely acknowledged that non-canonical RNA recognition elements abound in protein sequences ,66, and Another limitation is our inability to conclusively show the RNA binding capacity of SETD1B. Although our experiments consistently indicate a lack of robust RNA binding activity, we cannot conclude unequivocally that the RNA binding capability is completely missing in the case of SETD1B.