Echerichia coli. This may accelerate fermentation processes with the potential to reduce production costs. Similarly, elevated levels of secondary metabolites might be produced. Evidence for this possibility comes from preliminary findings regarding the production of surfactin in Bacillus subtilis, which has functions similar to those of bacteriocins. The prerequisite for its potential biotechnological utility is a general characterization of 6S RNA in LAB.6S RNA is a regulator of cellular transcription that tunes the metabolism of cells. This small non-coding RNA is found in nearly all bacteria and among the most abundant transcripts. Lactic acid bacteria (LAB) constitute a group of microorganisms with strong biotechnological relevance, often exploited as starter cultures for industrial products through fermentation. Some strains are used as probiotics while others represent potential pathogens. Occasional reports of 6S RNA within this group already indicate striking metabolic implications. A conceivable idea is that LAB with 6S RNA defects may metabolize nutrients faster, as inferred from studies of Lactobacillales order. It laid the foundation for a bioinformatic characterization of common 6S RNA features. This covers secondary structures, synteny, phylogeny, and product RNA start sites. The canonical 6S RNA structure is formed by a central bulge flanked by helical arms and a template site for product RNA synthesis. 6S RNA exhibits strong syntenic conservation. It is usually flanked by the replication-associated recombination protein A and the universal stress protein A. A catabolite responsive element was identified in over a third of all 6S RNA genes. It is known to modulate gene expression based on the available carbon sources. The presence of antisense transcripts could not be verified as a general trait of LAB 6S RNAs.We provide a genomic annotation of 6S RNA throughout the Despite a large number of species and the heterogeneity of LAB, the stress regulator 6S RNA is well-conserved both from a structural as well as a syntenic perspective. This is the first approach to describe 6S RNAs and short 6S RNA-derived transcripts beyond a single species, spanning a large taxonomic group covering multiple families. It yields universal insights into this regulator and complements the findings derived from other bacterial model organisms.The online version contains supplementary material available at (10.1186/s12863-021-00983-2). Lactobacillales. Shared metabolic characteristics and evolutionary relationships have been used as common markers for the identification, classification, typing, and phylogenetic analysis of LAB species [Lactobacillus genus has led to frequent taxonomic rearrangements [Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. These groups share the ability to catabolize sugars for the efficient production of lactic acid [Generally Recognized as Safe (GRAS). Their biotechnological relevance is a result of the many beneficial features that can be exploited, for instance, as starter cultures in the food industry, mediating the rapid acidification of raw material [Streptococcus (S.) and Enterococcus [Lactic acid bacteria (LAB) constitute a genotypically, phenotypically, and phylogenetically diverse group of Gram-positive bacteria that belongs to the taxonomic order of the  species . During ngements . One reangements . Currenttic acid . LAB conmaterial , or as pmaterial . Neverthrococcus .LAB are usually exposed to a wide range of harsh stresses, both in industrial environments and throughout the gastrointestinal tract. This includes acid, cold, drying, osmotic, and oxidative stresses . SurvivissrS according to the original gene designation in Escherichia coli [Aquifex aeolicus, species that obtain energy through photosynthesis like Rhodobacter sphaeroides, as well as pathogens such as Helicobacter pylori [E. coli and Bacillus subtilis [Bacillales, a sister-order of Lactobacillales. 6S RNA is about 160-200 nucleotides in length and adopts a rod-shaped structure with an enlarged internal loop or bulge flanked by large helical arms on both sides [σ70 in E. coli and σA in B. subtilis) in competition with regular DNA promoters. This sequestration of RNAP alters the housekeeping transcription at a global level that is seemingly advantageous when facing numerous types of stress [B. subtilis), pRNAs can persistently rearrange the structure of 6S RNA to induce RNAP release, thus restoring regular transcription [E. coli have provided evidence that nutrients are metabolized faster in 6S RNA knockout strains than in the parental wild type strain [Over the last decades many small non-coding RNAs have been identified as key regulators in a variety of bacterial stress response pathways and in bacterial virulence –12. A prhia coli , 14. A 6hia coli , 16. Thir pylori . The dissubtilis , 21. Theth sides , 23. 6S f stress , 24, 25.cription –30. Stude strain , 31. FurL. delbrueckii, an industrial starter for dairy products, where a highly abundant ncRNA was reported [S. pyogenes [The importance of 6S RNA in LAB is indicated by studies that report its abundant expression as well as metabolic changes upon its knockout. However, specific 6S RNA analyses in this important group of bacteria are scarce or the studied ncRNA was not recognized as 6S RNA. It is annotated only in about half of all LAB species analyzed in this study . Here, we identified it in about 91% of all known LAB species. An example is reported . Though pyogenes .Lactococcus lactis, the expression of 6S RNA has been linked to the carbon catabolite repression protein CcpA that binds to DNA at cis-acting sequences. These sites are called catabolite responsive elements (cre) [cre sites are degenerate pseudo-palindromes. In Bacilli a CcpA dimer was shown to bind to dsDNA upon association with the Ser46-phosphorylated form of histidine-containing phosphocarrier protein (HPr-Ser46-P) [L. lactis, 6S RNA levels were found to be increased during stationary and exponential phase in the presence of galactose or cellobiose, but not fructose, as the sole carbon source. CcpA repression is known to be relieved by galactose and cellobiose, but not by fructose. Moreover, 6S RNA was found to be about 3-fold upregulated in a CcpA-deficient mutant [cre element was identified upstream of the -35 region of its promoter. This indicates a potential interaction between CcpA and the 6S RNA gene that might be relevant for LAB in general. Notably, B. subtilis 6S-1 and 6S-2 RNA were not identified as a target for CcpA [For ts (cre) ; cre sitSer46-P) . In L. lt mutant  and a crfor CcpA .E. faecalis, a major opportunistic human pathogen, an additional transcript antisense to 6S RNA was detected [E. coli [B. subtilis or any other species to date (own observation). However, interdependent expression of genes around the 6S RNA locus was noticed for other bacteria, e.g. R. sphaeroides (Proteobacteria), where a salt stress-induced membrane protein gene on the opposite strand immediately downstream of the 6S RNA locus is expressed at elevated levels in a 6S RNA knockout strain [For detected . The aut[E. coli , B. subtt strain .Apart from these isolated findings, little is known about the sequence, structure, and physiological role of this regulatory ncRNA in the large and widely heterogeneous group of LAB. In this study, we have annotated and analyzed 6S RNAs systematically to lay a foundation for further investigations regarding its role in stress responses, metabolic processes and interactions with eukaryotic cells. Moreover, we investigated how wide-spread and universally relevant the species-specific observations stated above are for LAB (link to CcpA and the presence of an antisense transcript). This is also the first comparative study covering 6S RNAs in a set of taxonomic families, thus making it possible to draw more representative conclusions than in species-wise studies.Firmicutes including Bacillus subtilis, Bacillus halodurans, Clostridium acetobutylicum, Oceanobacillus iheyensis, and Thermoanaerobacter tengcongensis [Bacillus subtilis 6S-1 RNA than to its paralog 6S-2 RNA [We searched 6S RNA sequences in 1,092 genomes covering strains from all 371 sequenced LAB species publicly available in the NCBI database at the time of this study . While tongensis , only on6S-2 RNA .%). Additional File Weissella of the Leuconostocaceae family, represented with 13 species in our dataset. While only a weak 6S RNA locus was predicted in no more than four species of this genus, a significant amount of transcription could be shown for the syntenically conserved intergenic region downstream of rarA in publicly available RNA-Seq data for W. confusa and W. koreensis [Weissella species. See Additional File Weissella have a distinct singularity that was hardly picked up by our covariance-based search strategy. The typical rod-shaped structure with a central loop or bulge could not be confirmed for these non-canonical candidates.6S RNA was located in 1001 genomes  that includes Abiotrophia defectiva (Aerococcaceae) and Bavariicoccus seileri (Enterococcaceae). At the level of taxonomic families, the genus Vagococcus is significantly different from other Enterococcaceae (green). Similarly, Aerococcus is different from other Aerococcaceae. Lactobacillus is known to be the most heterogeneous genus within LAB [Lactobacillus 1-7, Pediococcus, brown).Figure thin LAB . This isLactobacillaceae species share a common subtree. Notably, a number of species from the Lactobacillus 6 group  and B. seileri (Enterococcaceae). In the 16S rRNA tree, the grouped Aerococcaceae are closely related to Carnobacteriaceae. The 6S RNA tree, in contrast, splits this group into two subgroups that are not closely related to Carnobacteriaceae.The phylogenetic reconstruction of LAB species based on a sequence alignment of selected 16S rRNA sequences is shown analogous to the 6S RNA-based reconstruction in Additional File rarA), that is found upstream of the 6S RNA locus in nearly all species, and the universal stress protein A gene (uspA), that is found downstream across almost all species except for Streptococcaceae and a few Aerococcaceae members.To characterize the genomic locus of 6S RNA in LAB, a synteny analysis was performed. Proteinortho  was usedrarA gene is part of a highly conserved family of ATPases found in prokaryotes as well as eukaryotes. Homologs are known as mgsA in E. coli, mgs1 in yeast (maintenance of genome stability A/1), and WRNIP1 (Werner interacting protein 1) in mammals. The encoded protein is involved in cellular responses to stalled or collapsed replication forks, likely by modulating replication restart [The upstream  restart –48.uspA gene belongs to a superfamily that encompasses an ancient and highly conserved group of proteins that are widely distributed among bacteria, archaea, fungi, flies, and plants. It was found to be induced during metabolic, oxidative, and temperature stress in Salmonella typhimurium [E. coli [uspA is known to be differentially expressed in response to a large number of different environmental stresses such as acid and salt stresses, starvation, exposure to heat, oxidants, metals, ethanol, antibiotics, and other stimulants - particularly within the genera Lactobacillus, Streptococcus, Enterococcus and Lactococcus [The downstream himurium  and link[E. coli . uspA istococcus –53.B. subtilis 6S-1 RNA [Aerococcaceae, Aerococcus, Carnobacteriaceae, Vagococcus, Enterococcaceae, Pediococcus, Lactobacillus 2, 3, 4, 6, 7, Streptococcus, and Lactococcus, see Additional File The consensus structure and sequence conservation of 6S RNA in LAB based on a mLocARNA  alignmen6S-1 RNA . The cenE. coli, R. spheroides and B. subtilis for which the start sites are experimentally proven. Fig. E. coli, A. aeolicus and R. spheroides, but in these cases at positions 4/5  of 172 representative 6S RNA sequences from LAB species and in relation to those of B. subtilis 6S-1 RNA as template , LAB pRNAs are closely related to pRNAs synthesized from ate Fig. . AlthougStreptococcus pneumoniae and Streptococcus pyogenes RNA-Seq data  located around position 136 that starts at the beginning of the L2 loop -tailing libraries . It is tata Fig.  and 4 [5provided . Here wer pylori . Notablycre site upstream of the 6S RNA promoter was reported in L. lactis, suggesting that 6S RNA expression is regulated depending on the available carbon source [cre site could be found in about one-third of all LAB species. Fig. cre sites at the 6S RNA locus. Additional File cre sites in the 6S RNA region. Additional File cre sites are most frequently found in Enterococcaceae but also in several Streptococcaceae and the Lactobacillus groups 6 and 7 , indicating that such transcripts are not a common trait among LAB.6S RNA transcripts were highly abundant in general , indicating active transcription in LAB grown under a wide variety of culture conditions and stresses. In line with previous findings , howeverB. subtilis 6S-1 RNA, we could not verify this for candidates of the genus Weissella. Nevertheless, we identified evidence for significant transcription of the respective loci in publicly available RNA-Seq libraries for two strains, see Additional File W. koreensis. Although no relevant match was found for W. confusa, the intergenic region downstream of the syntenically conserved rarA showed transcription that matched a 6S RNA transcript even though its putative secondary structure did not match a canonical 6S RNA. A TATAAT sequence is present at the -10 region of all candidates reported for Weissella, indicating the presence of a promoter. Similarly, a rho-independent terminator was predicted at the RNA’s proposed 3’-end. Thus, the presence of an actively transcribed 6S RNA-like transcript can be assumed. It will be interesting to investigate the functional consequences of this structural alteration.Here we identified the 6S RNA gene at a well-conserved genomic locus in LAB species that distinguishes this bacterial group from related bacterial clades. While the consensus secondary structure is typically canonical as described for cis-acting sequences. These are called catabolite responsive elements (cre) and are located either in the promoter region or within the coding sequence of the regulated gene [cre sites upstream of the 6S RNA promoter in about a third of all LAB species, mainly in Enterococcaceae but also in Streptococcaceae and some Lactobacillus subgroups. For Streptococcaceae and Enterococcaceae, the presence and regulatory importance of these cre sites has been reported and studied previously [L. lactis where 6S RNA is 3-fold upregulated upon deletion of the ccpA gene [Carbon catabolite control is a major regulatory mechanism for the modulation of metabolic activity of microorganisms to optimize carbon metabolism and energy use. It involves both carbon catabolite repression and activation. In most low-GC-content Gram-positive bacteria this regulation is mediated by the catabolite control protein A (CcpA) that binds to DNA at ted gene . CcpA cated gene . We founeviously , 60. On cpA gene .cre sites were also identified internally - in some cases in addition to the site at the 6S RNA promoter . In the latter, however, rarA is encoded in the opposite direction and known to be monocistronic [Streptococcaceae members encode a tRNA-Lys immediately downstream of 6S RNA, suggesting that both genes are part of the same operon. This assumption is supported by RNA-Seq data for S. pneumoniae . Therefore, the possibility of a common functional context remains vague at present.A general property of the 6S RNA locus in LAB is its location between the en genes . RarA isistronic . The RNAB. subtilis 6S-1 RNA. LAB 6S RNAs also share the syntenic proximity to rarA, located upstream of 6S RNA in nearly all LAB genomes. Many species additionally encode the UspA protein downstream of 6S RNA, which makes its identification comparably easy. The experimental evidence that was processed and analyzed in this study also demonstrated that 6S RNA is expressed in a multitude of LAB species across all taxonomic families and under varying culture conditions. This also highlights the important regulatory role of this ncRNA in bacterial metabolism, further supported by the frequent presence of cre sites in its promoter and coding region. The conservation of 6S RNAs makes it plausible to generally apply our findings to any LAB species in order to explore its biotechnological potential.Lactic acid bacteria include highly heterogenous species and the study of the role of non-coding RNA molecules, particularly 6S RNA, in the regulation of the response of these bacteria to different stress conditions has many potential applications, both within industrial and health contexts. The global transcription regulator 6S RNA is present in nearly all species and well-conserved throughout this group. It generally resembles the canonical form that is well described for Lactobacillales order according to the NCBI taxonomy classification  [Enterococcus faecium for example comprises 1109 genomes/subspecies. Fifty-one out of these are marked as “Complete Genome” and were thus considered in the present work. Lactobacillus fuchuensis is represented with three genomes out of which the most complete assembly is marked as “Chromosome” that was thus considered, and so on. Additionally, we added 13 strains that were characterized by our institute (CERELA-CONICET) even though they did not meet this criterion. Species with yet unclear specific names (sp.) were neglected. A total of 1,092 genomes were considered in this study. An overview of the genera analyzed here can be found in Table ftp.ncbi.nlm.nih.gov from the NCBI database [Several thousand genomes representing 576 species that cover 48 genera were listed as part of the 09/2018) . In ordedatabase .Table 1et al., and from the Rfam seed sequences for the 6S/SsrS RNA family  to cover the currently known 6S RNAs [−30 was applied. Previously not annotated 6S RNAs were identified with a covariance-based search performed with INFERNAL (v1.1.1) [−8 was applied. In this case, a primary hit was found in most species while unexpected secondary hits were rare and could be judged manually in later stages. Overlapping hits were joined. Hits were found in 973 out of 1092 genomes. Redundant sequences were merged to a single representative sequence resulting in 330 unique sequences that were aligned using Clustal Omega (v1.2.1) [Agitococcus lubricus, Lactococcus fujiensis, Facklamia hominis, Pediococcus damnosus, Lactobacillus babusae, Pediococcus cellicola, Lactobacillus cacaonum, Lactobacillus mucosae, Lactobacillus coleohominis, Lactobacillus gastricus, Lactobacillus equigenerosi, Lactobacillus malefermentans, Lactobacillus oryzae, Oenococcus oeni, Weissella kandleri, and Weissella koreensis. In total 172 representative 6S RNA sequences covering 947 genomes remained. This set was used for further analyses.Putative 6S RNAs encoded in LAB genomes were identified in multiple steps. A BLAST-based approach was performed using available 6S RNA annotations given in the NCBI RefSeq annotation, from Wehner  6S RNAs , 66, 67.(v1.1.1)  using th(v1.2.1) . Sequenc(v1.2.1) ) as wellupsA and/or rarA homologs. Both are typically encoded in close vicinity to 6S RNA gene , a second search iteration was performed with a LAB-specialized covariance model that was build based on all canonical 6S RNAs identified before. The e-value threshold was reduced to 0.1 and all search heuristics were turned off (cmsearch –max). In addition, the correct genomic locus was ensured by only allowing hits within 2000 nt from Terminators were predicted using TransTermHP (v2.09) . An adapEscherichia coli K12 (GCF_000005845.2) and Bacillus subtilis 168 (GCF_000009045.1), which codes for two paralogs, 6S-1 and 6S-2 RNA  [All representative 6S RNA candidates were aligned using mLocARNA (v2.0.0RC8), a local structural alignment algorithm for RNA secondary structures . To locand BsrB) , 72. Thend BsrB)  and visund BsrB) , excludiThe transcription start of 6S RNA-derived pRNAs was determined based on the structural alignment mentioned above. Based on previously characterized transcription start sites in other bacteria , 55, 74,Bacillus subtilis 168 (GCF_000009045.1) 6S-1 RNA (BsrB) was added as an outgroup [The sequences of the 6S RNA candidates identified in the first round were clustered hierarchically based on their structured RNA motifs using RNAclust . This apoutgroup . The resoutgroup .−20 based on the 16S rRNA reference sequences provided by the NCBI database [16S rRNA sequences were identified using BLAST (v2.8.1+)  with an database . Redundadatabase . The 5’-database  using thdatabase .The amino acid sequences of ten protein-coding genes 5000 nt up- and downstream of the predicted 6S RNA locus were fetched from the NCBI database. Orthologous groups were predicted with Proteinortho (v6.13) . To avoicre sites was derived from experimental B. subtilis data [L. lactis data [cre motif using MAST [rarA gene. Hence, we did not expect binding sites further upstream to be relevant to 6S RNA. We used the dinucleotide distribution of the respective genomes as background for each e-value calculation. The default e-value threshold of 10 and p-value threshold of 10−5 was applied. The resulting motifs were separated in two groups: Upstream of the 6S RNA promoter and within the 6S RNA coding region as shown in Fig. The sequence motif for lis data  that alstis data  as showning MAST . TypicalAvailable RNA-Seq datasets for LAB were located in the NCBISRA archive and downloaded on 12-112018 . In totaAdditional file 1 List of genomes and 6S RNAs (xls). List of LAB genomes used in this study including tax annotation, assembly status, location of the predicted 6S RNA.Additional file 2 Full 6S RNA phylogeny (pdf). Sequence- and structure-based reconstruction of 6S RNA phylogeny in LAB including the annotation of species with located cre sites. Full taxonomic resolution of Fig. Additional file 3 16S rRNA phylogeny (pdf). Phylogenetic reconstruction of LAB 16S rRNA.Additional file 4 Full genomic context of 6S RNA in LAB (pdf). Full genomic context of 6S RNA in LAB. Full taxonomic resolution of Fig. Additional file 5 6S RNA grouped consensus alignment (pdf). Folded consensus structure of the 6S RNA groups analogous to Fig. Additional file 6 RNA-Seq results (pdf). Visualization of RNA-Seq libraries mapped to the respective 6S RNA loci.Additional file 7 Predicted cre site motifs (xls). Predicted cre sites sequences and positions relative to the 6S RNA start site.Additional file 8 6S RNA evidence in Weissella (pdf). RNA-Seq data, genomic context and sequences of putative 6S RNA loci in Weissella.


Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a responsive organism by exogenous RNA. While exogenous RNA transfer between organisms of different kingdoms of life have been unambiguously identified in nature, our understanding of the biological significance of this phenomenon remains obscure, particularly within an evolutionary context. During the last decade multiple reports utilizing various mechanisms of natural eRNAi phenomena have been attempted to develop new agricultural traits and products including weed, disease and insect control. Although these attempts yielded mixed results, this concept remains extremely attractive for many agricultural applications. To better utilize eRNAi for practical applications, we would like to emphasize the necessity of understanding the biological significance of this phenomenon within an evolutionary context and learn from nature by developing advanced tools to identify and study new cases of exogeneous RNA transfer and eRNAi. In this opinion article we would like to look at the exogeneous RNA transfer from an evolutionary perspective, propose that new cases of exogeneous RNA transfer still remain to be identified in nature, and address a knowledge gap in understanding the biological function and significance of RNA transfer. We believe such approach may eventually result in a more successful use of this phenomenon for practical applications in agriculture. Regulatory RNAs (RegRNA) form a diverse class of non-coding RNA molecules in eukaryotic and prokaryotic organisms that regulate gene expression through a sequence-specific interaction with RNA or DNA. The significance of many classes of regRNAs in regulating the expression of complex networks of genes in organisms during development or in response to stresses has been described in numerous publications and reviewed elsewhere . The mosC. elegans [E. coli is taken up by C. elegans and loaded into the RNAi machinery and down-regulates the che-2 gene thus impairing the nematodes’ ability to find food [In addition to regulation of endogenous gene networks within an organism, another phenomenon that draws the attention of many biologists can be described as the regulation of gene expression in a receptive organism by exogenous RNA. Such sequence-specific regulation in a responsive organism by exogenous RNA is usually referred to as inter-kingdom or cross-kingdom RNA and/or environmental RNAi (eRNAi). The eRNAi term that in our opinion reflects a broader scope of this phenomenon and will be used in this publication to describe any cases of exogenous regRNA transfer in nature or designed and engineered in a laboratory. The first reported case of eRNAi was a regulation of gene expression by ingestion of exogenous dsRNA or bacterially-expressed dsRNA by  elegans . It was ind food . A growing number of reported examples of eRNAi describing RNA transfer between plants and microorganisms, plants of different species, and between plants and herbivorous insects can be found in several reviews ,10. The Botrytis cinerea delivered small RNAs into plant cells silencing host immunity genes, while the host plant delivered sRNAs to Botrytis to target fungal genes and attenuate fungal pathogenicity [Bremia lactuca resulting in significant reduction in fungal growth and sporulation [Plants occupy a unique place in the Earth’s ecosystem by continuously interacting with numerous pathogenic or symbiotic microbes and herbivorous invertebrates, and thus forming the base of the food chain pyramid. This constant interaction creates numerous opportunities for an RNA transfer mechanism as a communication or regulatory signal between plants and biotic environment to evolve. Regulation of gene expression by exogenous RNA delivered to plant cells by gray mold during this plant–microbe interaction is one of several compelling examples of eRNAi . Plants genicity . A practrulation . Howeverrulation . The lacDiabrotica virgifera virgifera LeConte) resulted in systemic down regulation of target genes and insect death [While the bidirectional eRNAi between plants and fungi relies on intimate cellular contact , the actct death . This wact death . Plant-dct death . At the ct death . Even wict death . This inNicotiana benthamiana plants overexpressing GFP, a process that seems to be dependent, at least in part, on the size and abundance of the siRNA effector as well as on the relatively high expression of the target gene [Inspired by examples of eRNAi found in nature and several cases of engineered eRNAi, there is increasing interest in leveraging the eRNAi for a broader range of practical applications in agriculture utilizing either plant incorporated or topical approaches. Plants have endogenous mechanisms to amplify regRNA signals and, in some cases, spread it systemically to distant cells or tissues  and thisget gene ,23,28,29In order for exogenous regRNA to achieve a noticeable regulatory effect in heterologous organisms, the regRNA not only needs to be efficiently taken up by recipient cells in a biologically relevant concentration in the host, but also must be protected from intracellular degradation and be compatible with host RNA regulatory biogenesis pathways and silencing machinery. Therefore, in addition to exogenous regRNA bioavailability, the differences in compatibility with host machinery, including efficient loading into the host silencing complex along with systemic spread, can also be factors contributing to the variability in response to eRNAi. Several published examples of bidirectional plant fungal eRNAi demonstrated the importance of exosome-based active delivery of fungal sRNAs . LearninIn nature, plants co-exist with a diverse set of microbes such as archaea, bacteria, fungi, protists and viruses forming a complex consortia called the “holobiont”. In such consortia where holobiont members cohabitate in close proximity for millions of years, co-evolution likely results in developing mutualistic, commensalistic or pathogenic interactions ,31. UnliSeveral reported examples mentioned above and reviewed elsewhere suggest a diversity of forms and mechanisms of exogenous regRNA transfer as outlined in C. elegans and some insect species, long dsRNA uptake is driven by specific mechanisms such as SID2 and clathrin-mediated respectively [Sclerotinia sclerotiorum [In ectively ,41. Whilrotiorum . Recent rotiorum ,44 also Apart from downregulation of target genes, exogenous RNA may trigger receptor mediated regulatory signaling, as in case of dsRNAs inducing pattern-triggered immune signaling , which iThe more we understand the evolutionary trajectory of eRNAi cases the more we will understand the mechanisms and biological function of this process in different organisms. This knowledge of native mechanisms can be leveraged to develop practical applications, including crop protection from insect pests, microbial pathogens and weeds, plant adaptation to abiotic stresses, promoting plant-microbe symbiotic interactions, and other possibilities aimed at improving agriculture. Significant technical challenges remain to demonstrate unambiguously the functional role of exogenous RNA in heterologous organisms, especially when the recipient/host has limited exogenous RNA uptake capacity and lacks amplification and/or systemic spread of the RNA regulatory signal. In such cases, a phenotypic effect or impact on the host is difficult to measure due to the temporal and localized nature of the response and challenges in identifying target cells and gene products or affected pathways. Elucidating knowledge that regRNA transfer occurs but does not result in a biologically meaningful effects could still add value to the understanding of the evolution of regRNA, exogenous RNA mobility, uptake and eRNAi. Expanding our knowledge of regRNA biogenesis and function in donor and recipient organisms, increasing the amount of available genomic and transcriptomic data and onboarding advanced detection techniques would allow us to identify currently undetected cases of eRNAi by specific signature marks of exogenous regRNA processing or association with host RNA regulatory machinery. These approaches also require caution in the interpretation of results since because of the high sensitivity of next-generation sequencing technologies and the possibility of cross contamination. Careful evaluation of the most appropriate techniques or combination of methods and unbiased interpretation of results are critical steps to distinguish between false positives and true eRNAi as well as to minimize false negatives and missing true cases of eRNAi. Advanced sRNA sequencing approaches such as degradome sequencing, cross-linking immunoprecipitation and single cell RNA sequencing in combination with laser captured microdissection, microfluidic-based cell sorting, extracellular vesicle purification and new bioinformatic tools relying on extensive databases could help to identify new true cases of eRNAi and better understand the evolution of eRNAi in plants and interacting organisms. Significant progress has been made studying extracellular RNA as cell-to-cell and long distance signaling in various organisms ,47 and sAs the aforementioned techniques have become more broadly available and affordable in recent years, their application to eRNAi studies may help us to conduct large-scale surveys of eRNAi events not only in the laboratory but also under natural environmental conditions. One example of such a survey could be a sampling of herbivorous insects from a natural feeding environment and conducting sRNA sequencing analysis for the presence of exogenous plant-derived RNA within insects and confirmed to be processed by insect RNAi machinery into insect siRNAs. Mapping the data to appropriate plant and insect genomes and transcriptomes  similar


Lying stalls for dairy cattle are designed to maintain cow hygiene, reduce labor associated with bedding maintenance, and provide cows with a comfortable place to lie down. These considerations can conflict: stall features that, e.g., reduce manure contamination of bedding can make the stall less comfortable, explaining why cows prefer lying in more open spaces. We developed an “alternative” lying area in which traditional freestalls  were modified to create larger areas, and flexible stall partitions were included to help maintain cleanliness. We assessed cattle lying behaviour, including lying postures, in this alternative pen compared to both traditional freestalls and an open pack. Not surprisingly, cleanliness was higher in freestalls, but the alternative pen offered substantial improvement in cleanliness over the open pack. There was little difference in postures associated with lying positions (such as lying with limbs outstretched) between the open pack and alternative pen, and both offered greater limb extension compared to freestalls. We conclude that this type of alternative pen can provide producers with the opportunity to improve comfort compared to freestall housing and improve cleanliness compared to housing in an open pack. Modern freestall barns for dairy cattle have been constructed with considerations for dairy cow cleanliness; partitions and other stall features such as neck rails are designed to reduce manure contamination of bedding and decrease farm labor. However, cows prefer to lie in more open spaces, including on bedded packs and pasture. We created an “alternative” housing area by modifying a traditional freestall pen and including flexible partitions to create larger lying areas. We assessed cattle lying behaviour, including lying postures, in this alternative pen (ALT) compared to an open pack (OP) and freestalls (FS) with different stocking densities. We also assessed levels of manure contamination across systems. Cleanliness was highest in FS, but ALT provided substantial improvement compared to OP. Cattle spent more time lying down in OP and ALT compared to FS. There were few differences in postures (such as lying with limbs outstretched) between OP and ALT, but cows in both of these systems more often lay in extended positions compared to when they were housed in FS. Housing in OP and ALT was associated with reduced perching for cows with high body weight; perching has been linked to an increased prevalence of both hoof lesions and lameness. Thus, alternative lying areas can offer a solution for producers seeking to provide cattle with the advantages of a more open lying area, while improving hygiene relative to an open pack. Modern indoor housing systems for dairy cattle are designed to help keep the animals and facility clean with minimal labour. Stall partitions and other design features of freestalls modify cow behaviour to discourage defecation onto the bedding surface (see ). ReduciWhen provided a choice between open packs and freestalls, dairy cattle show a strong preference for more open spaces for both standing and lying . Dairy cThe primary aim of the current study was to investigate differences in lying behaviour, including lying postures, in dairy cattle tested in different housing systems . Given the consequences of stall design on fecal soiling of the lying surface, a secondary aim was to quantify the effect of these different housing systems on bedding cleanliness. As the amount of space available per cow may affect both outcomes, and treatments differed in space available, we tested the freestall treatment at two stocking levels, fully stocked and stocked at 50% .Freestall, 50% stocking (FS1): Animals were housed in a conventional freestall pen fitted with two rows of six stalls : Housing and management for this treatment were as described for FS1, but the pen was stocked at 100% . This stocking density was achieved by housing two “groups” of six animals in the same pen.2 each. The pen was stocked with six cows . Feeding and management were as previously described.Open Pack (OP): All stall partitions and the neck rail were removed, with the exception of the partitions separating each row of stalls within the pen from the crossovers, leaving two “open pack” areas of 15.2 m2. This pen was stocked with 6 cows .Alternative Stall (ALT): Each row of six stalls was modified to create three large “stalls”, each 2.4 m wide . ModificAnimals were housed at The University of British Columbia’s Dairy Education and Research Centre in Agassiz, BC, Canada. In total, 48 non-lame, dry, pregnant Holstein cows  were included in the study. Cows were fed a total mixed ration (TMR) once per day, consisting of 30.1% straw, 26.75% grass silage, 21.86% alfalfa hay, 20.6% corn silage, and 0.7% dry cow mineral composition. The animals were fed at 08:30, and feed was pushed up three times per day. Water was provided through one self-filling trough in each pen, cleaned once weekly. Cows were enrolled 54 ± 9 d before expected calving date and blocked into eight “groups” of six based on dry-off date. Individuals in each group were dried off together and moved to their respective experimental pen. To facilitate individual identification, animals were marked with unique symbols using hair dye two days prior to their move to the experimental setup. The treatments were as follows:Each of the eight groups was tested for a seven-day period under each treatment condition. The order in which the treatments were presented to each group was randomized, with the constraint that an equal number of groups began and ended with the control (FS2) or an alternative housing area (OP or ALT). The order of treatments across the six groups is shown in Three cameras were mounted over the experimental pen: two dome cameras  were mounted over the feed alley and the lying area closest to the feed bunk. One camera  was mounted over the rear alley approximately 2.4 m from the window. Video was recorded continuously, and a red lamp  was used to facilitate nighttime video recording. We developed an ethogram  to measuStall cleanliness was calculated twice daily for the last two days of each treatment period. A 1 m × 1.6 m grid, comprised of 160 10 cm × 10 cm squares (as described in ), was plThe total number of soiled squares from each flooring area was summed across the two observations within a day and then averaged across the two observational days to provide an aggregate measure of lying surface cleanliness. Initially, the area underneath the plastic piping in ALT was not included in cleanliness scoring. However, during testing, we observed that cows occasionally extended limbs underneath this piping, so this flooring area was included beginning with the fourth group of cows; for this reason, data from Groups 1–3 in ALT were excluded from the analysis of stall cleanliness. These analyses were performed using SPSS  and GraphPad Prism . Absolute agreement between observers for cleanliness scoring was assessed using the interclass correlation coefficient . We calculated separate ICC values for the bedded pack  and for the freestalls  due to slightly different processes used in obtaining the cleanliness measurements in each of these areas. Three overall Cohen’s kappa coefficients were calculated for behaviour (lying or standing), posture, and head position  and figures were generated using GraphPad Prism . Data were first studied graphically and descriptively using PROC Univariate and PROC Freq to gain familiarity with the frequencies and distributions of each variable.We created 2 main behavioural models, with Average Time Spent Perching (per 24 h) and Average Time Spent Standing (per 24 h) each serving as outcomes and Treatment as the predictor of interest. In addition, to explore time spent lying down, we created 4 models to evaluate whether Treatment could predict the amount of time that cows spent lying in certain positions : Lying down with no limbs extended (corresponding to “None” in the ethogram), Lying down with both hind limbs extended (“H2” in the ethogram), Lying down with the head curled (“Curled” in the ethogram), and Lying down stretched out. This final category represented a combination of several lying behaviours recorded in the ethogram; specifically, a cow was recorded as Lying down stretched out if she was either in a full lateral position (“Full” in the ethogram) or if she had both hind legs extended (H2 in the ethogram) in addition to resting her head on its side with neck outstretched (“Down” in the ethogram). These behaviours were combined because “Full” was rare, and the simultaneous occurrence of “H2 + Down” corresponded to a similar space allocation to “Full”.We generated a correlation matrix of all potential predictor variables to evaluate the assumption of multicollinearity using r ≥ |0.70| as a threshold. Body Weight (BW) and Body Condition Score (BCS) were highly correlated (r = 0.77) and were thus not included simultaneously in any model. Multivariable mixed linear models for the following outcome variables were constructed using PROC Mixed in SAS: Average Time Spent Perching, Average Time Spent Standing, Lying down with no limbs extended (“None”), Lying down with both hind limbs extended (“H2”), and Lying down with the head curled (“Curled”). The variables Treatment , and Parity (categorized as 1st or ≥2nd), were included as fixed effects in all models regardless of significance level. Other potential fixed-effect predictors were BCS, BW, DCC, and all relevant two-way interactions were retained in the model if significant, using a manual backwards stepwise elimination procedure. Cow ID nested within Group was included as a random effect in all models. Planned contrasts were conducted using the Contrast statement to compare: (1) alternative housing types (ALT and OP) to freestall housing (FS1 and FS2); (2) half versus fully stocked pens (FS1 versus FS2); and (3) open to semi-open housing (OP versus ALT). If an interaction was present between Treatment and a continuous variable, the variable was retained as continuous in the model but was categorized for further exploration with planned contrasts.Due to low counts in the outcome variable Lying down stretched out, a mixed Poisson regression model for over-dispersed data was developed using PROC GLIMMIX in SAS. Again, Treatment and Parity were included as fixed effects and Cow ID nested within Group as a random effect. Planned contrasts were conducted as previously described.By means of PROC Mixed, a mixed linear model was created to evaluate the cleanliness of each housing type. The outcome variable was the proportion of available flooring area contaminated by manure per cow (based on the number of cows present in the pen at a given time). Treatment was considered as the fixed effect of interest, and Group was included as a random effect. Data were averaged across two days of observation, with the exception of one case in ALT in which only one day of data was available. Additionally, due to a data entry error, Week 6 in OP could not be included in the analysis.Denominator degrees of freedom were calculated using the containment method. Because two different groups were present at one time in the FS2 treatment, and hence were not independent, a manual adjustment was made to the denominator degrees of freedom to reflect an accurate number of unique observations. Of a total of 28 observations, only 24 were unique; thus, the denominator degrees of freedom were reduced from 17 to 13 using the “ddf” = option in SAS. Planned contrasts were conducted using the contrast statement.One of the outcome variables (Average Time Spent Perching) required a cube-root transformation to achieve normality of model residuals. We selected models with the lowest Akaike information criterion , with the stipulation that the variables Treatment and Parity were always retained. A variance components (VC) covariance structure was selected for all models, as no model improvements were identified when the structure was altered. For linear models in which the outcome was a proportion, all predicted values were in a plausible range .The ICCs  for cleanliness scoring were 0.98  in the bedded pack and 0.99  in the freestalls. Although only two raters were used, the lower bound of the 95% CI for both ICCs was above 0.90, indicating excellent agreement . A total of 13,825 observations from each of the two observers was available for assessing interobserver reliability in behaviour, posture, and head position. The observations spanned 8 days and included observations from 24 animals. There was near-perfect agreement for behaviour (kappa (95% CI) = 0.99  according to criteria outlined by Landis and Koch , and thep = 0.02) in alternative housing systems  compared to freestalls . BCS was a predictor of standing time, such that standing decreased as BCS increased   did not differ between treatments, with cows spending an average (±SE) of 43% (±0.9%) of their time standing; however, planned contrasts revealed increased lying time , in half versus fully stocked freestalls (FS1 versus FS2) (p < 0.001), and in OP compared to ALT (p = 0.019). For low BW cows , the only reduction in time spent perching was between OP and ALT (p = 0.002) (There was an interaction between treatment and BW on Perching (F= 0.002) .3,277 = 2.79, p = 0.041, p < 0.001). This difference was no longer evident for cows in higher parities. Both 1st and ≥2nd parity cows spent more time in this posture in freestalls (FS1 and FS2) compared to alternative housing areas (ALT and OP) and in half versus fully stocked pens (FS1 versus FS2) (p < 0.001).There was an interaction between treatment and parity on the amount of time cows spent lying down with no limbs extended , as did cows with higher BCS . There was also an effect of treatment , in half versus fully stocked freestalls (FS1 versus FS2) (p < 0.001), and in ALT compared to OP (p = 0.033).Cows in parity 2 or higher spent more time lying with both hind limbs extended . There was also an effect of treatment on time spent in this posture  and in half versus fully stocked freestalls (FS1 versus FS2) (p = 0.001). There was no difference between the ALT and OP treatments  and in half versus fully stocked pens (FS1 versus FS2) (p = 0.014). There was no difference between ALT and OP treatments.The number of times that cows were observed lying down in a stretched-out position also differed between treatments  in freestalls (FS1 and FS2) versus alternative housing areas , and in semi-open versus open housing areas .The percentage (±SE) of flooring area per cow contaminated with manure varied with treatment , and in half versus fully stocked pens. However, first parity cows assumed this position more often in ALT compared to OP. This finding was somewhat surprising, as we might expect less behavioural plasticity in older cows habituated to freestalls. The current results suggest that factors associated with age, such as larger udders or increased BW, may make these postures especially important.Not surprisingly, cleanliness level was highest in freestalls: hygiene is a main factor influencing the design of these systems . More inThis study documented the effects of several housing methods on lying time and specific lying postures associated with cow comfort. We found that postures such as lying with outstretched limbs, or with the neck curled, were least frequently observed in freestalls. Importantly, these postures were not compromised in the alternative housing pen compared to the open pack. Although stall cleanliness was highest in freestalls, the alternative system offered improvements in cleanliness compared to the open pack. Thus, we conclude that a modified lying area, such as that provided by the alternative treatment, can offer improved cleanliness in comparison with an open pack, and improved cow comfort relative to freestall housing.


Due to the high exposition to changing environmental conditions, bacteria have developed many mechanisms enabling immediate adjustments of gene expression. In many cases, the required speed and plasticity of the response are provided by RNA-dependent regulatory mechanisms. This is possible due to the very high dynamics and flexibility of an RNA structure, which provide the necessary sensitivity and specificity for efficient sensing and transduction of environmental signals. In this review, we will discuss the current knowledge about known bacterial regulatory mechanisms which rely on RNA structure. To better understand the structure-driven modulation of gene expression, we describe the basic theory on RNA structure folding and dynamics. Next, we present examples of multiple mechanisms employed by RNA regulators in the control of bacterial transcription and translation. In the course of evolution, bacteria have developed many mechanisms enabling a rapid adaptation to environmental conditions via instant regulation of gene expression. Among them, the fastest and most efficient is the employment of regulators based on modulation of the RNA structure. It is possible due to the intrinsic RNA’s ability to fold into sophisticated structures through the base pair interactions among distantly located regions as well as by pairing local nucleotides into hairpins. These structures are highly dynamic and can comprise motifs as simple as base-paired double-stranded regions that form in trans or cis ,2, and aDynamically changing RNA structure is involved in the control of the majority of gene expression steps, from transcription, through translation, up to RNA decay ,5. In prThe topic of RNA structures in different cellular processes has been covered in several excellent reviews ,12,13,142+, K+) and complex metabolites , as well as binding structural, processing, and signaling proteins [The versatility of RNA in forming both simple and complex structures allows it to engage in diverse interactions with other molecules, ranging from base-pairing with small RNAs to forming binding pockets for cations . The binding of the F-, in a narrow concentration range, stabilizes this structure and triggers the conformational changes in the expression platform . An exam binding .RNA thermo-sensing structures, called RNA thermometers, undergo temperature-induced conformational changes or processing transformations to control the gene expression in bacteria. We can distinguish two mechanisms of thermoregulation: cis-acting translation initiation regulation and trans-acting antisense binding D. Cis-acipbA in E. coli [An example of the mechanism of a cis-acting switch is ROSE (Repression Of heat Shock gene Expression), present in the 5′ UTR in Gram-negative bacteria. ROSE controls the translation of the expression of small heat shock genes, such as the inclusion body binding protein A gene  E. coli . RNA the E. coli . In thei E. coli  showed t E. coli .E. coli. A higher expression of RpoS is observed at 25 °C, causing the activation of RpoS-dependent promoters at lower temperatures. Expression of RpoS is regulated by the 85-nucleotide small noncoding RNA DsrA, the concentration of which is also higher at 25 °C. DsrA RNA takes two forms: the primary transcript F form, which is then further processed into a 60–61-nucleotide T form, lacking the 5′-region interacting with the rpoS mRNA. Since the primary transcript (F form) is more stable at 25 °C, the interaction with the leader sequence of the rpoS transcript is enabled. By binding the antisense RNA (5′ region of DsrA), the disruption of the base pairing in the flanking SD region occurs [rpoS mRNA is accelerated by the Hfq protein binding, which stabilizes the tertiary interactions of those RNAs [rpoS mRNA changes the conformation and activates the translation process, resulting in the increased expression of RpoS at low temperature.The documented example of thermoregulation by trans-acting antisense binding is the RNA polymerase sigma factor S (RpoS) regulation in n occurs . The paiose RNAs . The rpoTranscription termination plays an important role in the regulation of gene expression, allowing recycling of the RNA polymerase (RNAP), preventing transcription of the neighboring genes, and collisions with other transcriptional complexes. In bacterial genomes, two different modes of the termination process are distinguished. The first is based solely on the interaction between RNAP and DNA-template sequence , while the second involves cooperation with specific factors, such as Rho or Mfd proteins . In this review, we will only briefly describe factor-dependent transcription termination mechanisms as they are not directly regulated by RNA secondary structures.Intrinsic termination is associated with the formation of the G-C-rich hairpin structure (terminator hairpin) on the nascent RNA molecule, within 7–9 nucleotides (nt) upstream from the RNA 3′ end ,50. ImpoE.coli, B.subtilis, and only a few other species, therefore leaving a huge variety of different microbial lineages relatively unstudied in terms of transcription termination regulation. As an example, Ahmad et al. [It is still under debate as to what extent the features, described above as essential for efficient intrinsic termination, are conserved across all bacteria. Most of the research is based on d et al.  showed td et al. .Roberts  in his rAlthough, in general, the intrinsic termination does not require any additional factors to cause the transcription termination, some of the factors have the potential to increase or decrease the efficiency of transcription termination. The most widely studied elongation factors, NusA and NusG, are relatively conserved across bacteria and archaea. They both stimulate the termination process by extending or introducing new RNAP pauses respectively, and increasing RNAP processivity ,54,55,56Mollicutes, Cyanobacteria, and some species within Clostridia and Bacilli classes [E. coli. Washburn and Gottesman in their review [Rho- and Mfd-dependent termination processes belong to the distinct class of transcription events. In contrast to the intrinsic termination, those mechanisms are dependent on auxiliary factors such as Rho or Mfd translocases. Mfd-dependent termination is devoted to the recycling of the stalled elongation complexes that otherwise prevent DNA replication. Mfd binds to the RNAP and the upstream DNA sequence and initiates the translocation of the stalled complex, leading to the release of the nascent RNA molecule and the disassociation of the RNAP from the DNA template strand . Rho is  classes . Rho is r review  state thr review  declare r review ,59. The r review ,57,58,59r review ,58. Tranr review .Some of the transcription termination mechanisms existing in bacteria can terminate the process conditionally, in response to environmental factors. Here, we list the most prominent examples of such mechanisms, also providing several important reference articles, allowing the reader to further explore the topic in detail.Riboswitches are RNA structures allowing bacteria to conditionally terminate transcription of the controlled gene(s) in response to binding a small molecule or ligand  operon in E. coli can be an example of transcription regulation by ribosome stalling  in Salmonella enterica. On the other hand, a thermosensor with a switching mode was found in enterobacteria phage λ, where it changes the structure from A to B. When the temperature increases, structure A stabilizes and prevents the synthesis of cIII protein, promoting bacteriophage into the lytic cycle, and structure B enables translation of this protein. Thermosensors can also be triggered by temperature decrease, e.g., in control of the cold-stress response in E. coli. In this case, the cold-induced structural rearrangement causes the release of RBS from the stem, activating the synthesis of cold shock protein A (CspA) [Thermosensors and riboswitches are the most ubiquitous representatives of RNA structures regulating translation in cis. Three classes of bacterial genes are under temperature-dependent control—virulence, cold shock, and heat shock genes. In translation, thermosensors act by sequestering RBS in a secondary structure that responds to temperature fluctuation D. They cbacteria ,71. OtheA (CspA) .Even more complex translational regulators are riboswitches. Similar to RNA thermosensors, they control the initiation step by modulation of the RBS structural availability, however, the process is induced by binding the small ligands. ,72. As aB.subtilis, wherein the presence of a glucosamine-6-phosphate (GlcN-6-P), the glmS riboswitch activates the self-cleaving ribozyme in the 5′ UTR of transcript coding glutamine-fructose-6-phosphate amidotransferase (GlmS). Next, the coding region of the mRNA is rapidly degraded by RNase J1 [E. coli, which can preclude the translation by sequestering the RBS sequence into the secondary structure and the same riboswitch can target mRNA for degradation by RNase E [Another interesting mechanism that involves riboswitches is the indirect regulation of the translation degradation of the mRNA. This phenomenon was discovered in Gram-positive bacteria, like RNase J1 ,73. Anot RNase E ,74.RNA secondary structure can modulate translation also via induction of the ribosome pausing and collision. Increased pausing leads to lower efficiency of protein synthesis since the time needed for a single translation is longer and fewer proteins will be synthesized from single mRNA within its cellular lifetime. There are few reasons for the ribosome pausing, including strong mRNA secondary structures during translation initiation and ribosome translocation. Since ribosomes possess the helicase activity and can unwind the secondary mRNA structures, the pausing during translocation is rather temporary. Such pauses can lead to changes in translation efficiency only when many ribosomes are translating the same mRNA, creating polysomes. The situation, when pausing takes place on a polysome leads to the queuing of the ribosomes. In extreme cases, collisions of the ribosomes can occur, which are recognized in bacteria by the RqcH, a protein that is a part of the ribosome quality control system and marks the nascent protein chain to degradation by the ClpXP protease .+). The same structure can be also formed by RNA (rG4s). A recent study showed that rG4s can be found in many bacteria species. Shao and colleagues revealed some more insights into the rG4s. They demonstrated that the distribution of these structures is species-specific and that rRNAs are less abundant in the form of quadruplexes. Moreover, they associated rG4s with important biological processes in E. coli and Pseudomonas aeruginosa, including regulation of metabolic processes and gene expression [Also, the higher-order structures can be involved in translation regulation in bacteria. G-quadruplexes (G4s) are G-rich structural motifs, originally discovered in DNA, that can fold into a minimum of two stacked G-quartets (four guanines connected by H-bonding) and are stabilized by the potassium ions . In case of lack of GlmZ, the SD sequence of glmS mRNA is occluded in the hairpin structure. To activate the translation of glmS mRNA, GlmZ binds to the RBS-masking hairpin acting as an anti-antisense element and thus releasing the RBS for ribosome binding. GlmZ levels are strictly regulated by other ncRNAs, including YhbJ and GlmY, which together enable complex homeostasis of GlmS expression [As mentioned earlier, ncRNA binding can also activate the translation. One of such regulatory processes, involving GlmZ and GlmY ncRNAs occurs co-transcriptionally taking advantage of the formation of intermediate structures of nascent transcripts. It was observed in pression ,72.E. coli (csrB and csrC is regulated by a two-component signal transduction system consisting of BarA and UvrY. Interestingly CsrA can indirectly influence these sRNAs’ transcription through the response regulator UvrY. CsrA is known to inhibit glycogen synthesis, peptide transport, synthesis of a biofilm adhesin, and activate cell motility, glycolysis, and acetate metabolism. Homolog molecules working in similar systems were found also in other bacteria species, for example in Pseudomonas fluorescens and Erwinia carotovora [Another type of translation regulation mechanism based on RNA structure is represented by a Csr system discovered in E. coli B. The throtovora ,77.The highly dynamic nature of RNA, together with a coupling of the transcription and translation processes, provides a wide range of possible regulatory mechanisms. Although our understanding of the role of RNA secondary structure in gene expression regulation constantly grows, some aspects are still unresolved. One of the most promising recent developments which can be used for such research is related to the establishment of high-throughput structural probing techniques, such as Structure-seq, DMS-MaP, SHAPE-MaP, PARIS, and others. Similarly, as the introduction of small RNA high-throughput sequencing revealed a plethora of novel small RNAs in bacteria, a massive application of such structure probing techniques can lead to the identification of novel RNA structure-dependent regulatory mechanisms.


Pseudomonas, Hfq recognizes repeats of adenine–purine–any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation–repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq–RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a ‘status quo’ staging point, providing nonspecific binding affinity, until Crc engages the Hfq–RNA binary complex. The dynamical components of the Hfq–RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base–phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq–RNA binding.The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In  A). The hexamer can bind RNAs via three surfaces   , 2, 3, 4Fig. 1surfaces , 8, commsurfaces , 10. Fursurfaces , 12. TheFig. 1B) .Figure 1Pseudomonas aeruginosa, Hfq was identified as a versatile contributor to metabolic regulation . Depending on availability, up to two additional Crc proteins can be recruited into the quaternary complex  simulations to explore the conformational variation of the Hfq–RNA binary complex and the higher order quaternary complexes formed with Crc. Our MD simulations utilize a set of carefully calibrated molecular mechanics models , 22 thatdynamic recognition. The substates associated with the dynamical ensemble may be important for the detailed mechanisms of the process of binding and unbinding  can undergo frequent syn/anti flips, before Crc binding. The anti and syn conformations are supported by Hfq via unique adenine-specific interactions in both positions. The frequency of the flips is lowered upon formation of the quaternary complex, that is, after Crc is bound, and the syn conformation becomes less favored. We also identify a previously unknown binding pocket at the distal side of Hfq which can weakly bind the N nucleotides of the ARN repeats in the absence of Crc. Finally, we suggest a potential assembly pathway in which the Crc initially recognizes an intramolecular RNA interaction in the amiE6ARN.Our results suggest that extensive equilibrium local dynamics indeed occur at the distal side interface of the Hfq–RNA complex and offer a concrete example of a conformational switch that can influence rates of translocation along a length of RNA. Namely, the first nucleotide in each ARN repeat . All ARN motif nucleotides in the positions “A”  and “R” , respectively, are bound in identically organized binding pockets, and their positions are clearly defined by H-bonds, base stacking, and van der Waals (vdW) interactions (A). The pockets exhibited highly similar behavior in simulations, and their protein–RNA interactions were all maintained with only reversible fluctuations –AA(N7) describes backbone amide nitrogen of residues Q33 in the individual Hfq chains forming H-bonds with N7 of RNA nucleotides A1, A4, A7, A10, A13, or A16. Wherever the behavior significantly differed among the repeats, we refer directly to the specific nucleotides by their residue numbering .The bound RNA in the quaternary complex consists of six ARN trinucleotide repeats while the Hfq itself is a hexamer B. All ARractions A. The potuations  and S2. umbering . To helpanti and syn conformations of the AA nucleobases. Most strikingly, the anti-AA and syn-AA nucleotides established interactions with the same amino acids. Structurally, this was possible by the N7 atom replacing the N1 atom as the H-bond acceptor and vice versa, whereas the second hydrogen of the N6 amino group was utilized as a H-bond donor . This water bridge is present in the X-ray structure of poly-A RNA bound to Hfq . In MD simulations, the K31(O)-AA(O4′) distance increased when AA flipped into syn, thus relieving the repulsion. The average simulation time between the flips differed significantly among the individual simulations and AA nucleotides, ranging from tens to hundreds of nanoseconds (B) was stabilized by a temporary formation of an intranucleotide H-bond, and the transition time of the flips was in the range of tens of picoseconds. In each simulation, several back-and-forth syn/anti flips were observed for every AA nucleotide with few exceptions  -AA(O4′) distance was shorter in the presence of Crc than in isolated Hfq–RNA (C), that is, Crc pushes the AA nucleotide deeper into the A pocket. This could affect the syn/anti balance as a tighter geometry is associated with the anti conformation. Flipping of the AA base into syn to relieve the K31(O)-AA(O4′) repulsion then becomes less favorable when Crc is bound. On the other hand, the penalty for the loss of the water bridge (B) upon flipping into syn remains the same in both systems. In conclusion, rather than simply blocking the flips, the Crc could also promote the anti conformation by subtly shifting the free-energy balance among multiple interactions within and around the AA binding pocket.The rmediate  for the rmediate  where alectively A 19). F. Fsyn/anwnstream . The synne suite  but not ormation . This inwith Crc . For bot Hfq–RNA C, that ir bridge B upon flA nucleotides in the anti conformation. In a quaternary complex where we flipped all the AA bases into syn before the simulation start , AA(N7), and GR(O6) atoms only when AA is in syn  interaction  or the Crc2 protein  , some of the adenines are in the syn conformation, and the study also revealed evidence of syn/anti flips  , 40 withti flips . Howeverllection , 43, 44.lusively . Our vislusively , 40 is csyn/anti states predicted by MD simulations may be influenced by potential mild force field imbalances, and thus, the results may not reach quantitative accuracy. However, we suggest that the simulations unambiguously predict that the Hfq A pocket can readily host A in syn conformation. The syn conformation, even if overpopulated by MD, could represent a transient (higher energy) binding pattern (syn conformation of A-pocket adenosines was significantly more populated when the succeeding R-pocket nucleotide was guanosine instead of adenosine (syn orientation or are able to capture the transition conformation (B) for binding.Populations of the  pattern  involveddenosine , suggestormation B for binA nucleobases and Crc directly promoting the anti conformation are formed only for the A1 and A13 . Despite this, our simulations of the quaternary complex revealed very few flips toward syn for these nucleotides when starting from the experimental structure where they all possess the anti conformation   . HoweverIn summary, we suggest that the “disordered” N-pockets weakly contribute to binding affinity of RNAs to the Hfq in a sequence-independent manner. The dynamical N-nucleotides are at the same time able to quickly establish interactions with other partners, which can already be sequence dependent.The cryo-EM structure of the quaternary complex  shows ex18 nucleotide and its 4BPh interaction toward the A3 phosphate might be the marker which, at least in case of the amiE6ARN mRNA, provided a specific binding site for Crc. The guanine-specific 4BPh interaction is the strongest base–phosphate interaction occurring in folded RNAs . In. In18 nuructures . We do nIn this study, we explore the dynamics of RNA recognition by a conserved and pleotropic RNA chaperone, Hfq. We used state-of-the art atomistic MD simulations to obtain high-resolution insight into local dynamics and substates associated with RNA binding to the Hfq distal site. We show that Hfq partly utilizes dynamic recognition of RNA substrates, a type of molecular recognition that is difficult to fully resolve in structural experiments. However, we suggest that dynamic recognition is likely an important contribution to structural mechanism by which Hfq engages RNA targets with adequate specificity while maintaining high RNA turnover rates in the cell. Upon presentation of a target RNA to effector molecules, such as Crc, this turnover is slowed down significantly, allowing for a downstream cellular response.via crystallographic symmetry. We have also used the X-ray structure of Escherichia coli Hfq bound to RNA poly-A octadecamer for simulations of the isolated Hfq–RNA system   as startA system . StartinOL3    and ff12e., OL3)  force fie., OL3) .A(OP2) and G29(O)–AR(O2′). Both of these H-bonds are present in all available experimental structures of P. aeruginosa Hfq bound to RNA-containing ARN repeats , HBfix + and Cl− atoms (In all simulations, the biomolecular systems were surrounded in a truncated octahedral box of SPC/E water molecules  with a ml− atoms .The systems built in tLeap were minimized and equilibrated according to the protocol extensively described in Ref.  utilizinThe cpptraj  was usedThe authors declare that the data supporting the findings of this study are available within the article and its This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Polycystic ovary syndrome (PCOS) is known as the most common metabolic/endocrine disorder among women of reproductive age. Its complicated causality assessment and diagnostic emphasized the role of non‐coding regulatory RNAs as molecular biomarkers in studying, diagnosing and even as therapeutics of PCOS. This review discusses a comparative summary of research into microRNAs (miRNAs) and long non‐coding RNAs (lncRNAs) that are molecularly or statistically related to PCOS. We categorize the literature in terms of centering on either miRNAs or lncRNAs and discuss the combinatory studies and promising ideas as well. Additionally, we compare the pros and cons of the prominent research methodologies used for each of the abovementioned research themes and discuss how errors can be stopped from propagation by selecting correct methodologies for future research. Finally, it can be concluded that research into miRNAs and lncRNAs has the potential for identifying functional networks of regulation with multiple mRNAs . This new understanding may eventually afford clinicians to control the molecular course of the pathogenesis better. With further research, RNA  may be used as biomarkers for the disease, and more possibilities for RNA therapy agents can be identified. We know that RNAs have regulatory functions in the way of protein production. It may follow that the disorders in protein levels that lead to many diseases could have first happened at the RNA level. Earlier changes could be diagnosed in the RNA level as an early diagnosis method for diseases with genetic traces. Recent developments in RNA immunotherapies, RNA vaccines and a vast field of RNA biomarkers are examples of their increasing role in modern medicine. The central role of RNAs in the transcriptome network makes them promising candidates as molecularly significant biomarkers of disease.RNAs vary greatly in roles, size, conformation and sequence. Non‐coding RNAs (ncRNAs) (not translated into proteins) with complex regulatory functions have gene and protein regulatory functions. Two main groups are miRNAs with about 22 nucleotides and long non‐coding RNAs (lncRNA).In this review, we first overview the prominent research groups working on miRNAs and PCOS, and then those working on lncRNAs. We follow with a discussion of combinatory and creative studies into the molecular basis of PCOS. We discuss how each research approach can be helpful on its own or when combined or integrated with other available methodologies. We also discuss the innovative methods and approaches that allowed leading research groups to gather key findings and share our insights into how these findings may determine future research into the molecular mechanisms and biomarkers for PCOS. This article also includes descriptive finding data for the found RNA biomarkers.22.1The functional molecular role of RNAs has been observed in many key processes, such as their enzymatic role in protein translation at ribosomes.in vitro has achieved recent worldwide prominence in synthesizing an RNA‐based COVID‐19 vaccine.Long non‐coding RNAs, on the other hand, are normally more than 200nt long and can have either linear or circular conformation. lncRNAs are core to many cellular functions such as gene regulation, transcription, chromatin modification and epigenetic regulation.2.2One of the major symptoms of PCOS is the presence of numerous small cysts in the ovaries. One method used to diagnose PCOS in the clinic is internal sonography of the ovaries.In general, there are two main approaches to validate a reliable biomarker for the disease. One is to use the potential of sequencing and blotting to differentially investigate a pool of possible RNAs and proteins in two populations of healthy and PCOS affected cells and patient blood samples or follicular fluid. From this pool, the most significant biomarkers are selected. The second is to investigate the molecular mechanism and biological significance of a known ncRNA biomarker for the syndrome.As far as finding new biomarkers is concerned, there is diversity but promise in the results. Che et alSeveral statistically significant miRNAs have already been identified as candidates for a clinical PCOS biomarker. The most statistically significant miRNAs included 381‐3p, 29a‐5p, 93, 320, 3188, 612, 509‐3p, 547‐3p, −5p, 20‐3p and −5pn . Not surprisingly, drugs can alter the levels of miRNA in PCOS conditions as well. Udesen et al.With miRNAs compromising the greatest share of RNAs in exosomes, exosomal miRNAs are a sub‐group with demonstrated scientific significance for PCOS diagnosis,in vitro, which can make various therapeutic and diagnostic agents. The COVID‐19 pandemic is also increasing the speed of the process of RNA synthesis in diagnostics, vaccine and therapeutic industries. These methods and the complete set of bioinformatics enabled many groups, as summarized in Table Numerous technologies recently boosted the use of RNAs in modern medicine. Modern sequencing, mRNA synthesis and modern genome editing are the most prominent. Recent progress in sequencing has enabled us to perform rapid descriptive transcriptome‐wide analysis.2.3p‐value or overall statistical significance, the fold of change in condition, specificity of the change to the condition, sensitivity of the change to special symptoms and the molecular role of the biomolecule.Efforts for better insight into the competing endogenous(ce)RNA networks affecting PCOS have also led to higher knowledge of the molecular networks and functions in the endocrine and female reproductive system overall. Overall, the literature indicates that lncRNAs play a key role in making up endogenous networks of bilaterally effective relationships between proteins, nucleic acids and various other types of RNAs (mainly mRNAs and miRNAs).Multiple groups have exploited the Gene Ontology (GO) and pathway analysis with Kyoto Encyclopedia of Genes and Genomes (KEGG) to mine out substantial relationships between their discovered RNA biomarkers for PCOS and important related biological functions.Zeng et al.Huang et al.in vitro and then in vivo. There are also other studies focused on circular RNAs in PCOS. Both Che et al. and Ma et al., have performed a descriptive study on circRNAs present in cumulus cells, which is of high prominence in PCOS research, given the role of cumulus cells in oocyte maturation and the role of circRNAs in cellular interconnection.Circular RNAs are the type of long RNAs with a covalently closed‐loop shape and may form coding or non‐coding genes. Hence, they can be both gene information carriers for proteins as well as gene regulators.via connexin 43 in granulosa cells and Bouckenheimer et al. showed that oocyte lncRNAs could have roles in chromatin remodeling, cell pluripotency and also in the early development of the embryo. They did so by analyzing the differential lncRNA expression profiles in human oocytes and cumulus cells. A manner pretty widespread among groups, as a list of prominent studies regarding lncRNAs and PCOS with similar methods, is available in Table As far as the type of biopsy for the biomarker is concerned, there are numerous groups working on blood serum, follicular fluid or cells of the ovarian tissue. Also, in order to trigger the attention on new biomarkers for the disease, studies on cell lines can also be favorable, especially when the line is correlated to a special condition of the disease, like that for KGN as performed by many groups.3Polycystic ovary syndrome (PCOS) is a disorder in which women of reproductive age suffer, causing disorder in levels of hormones. Having direct relation with infertility issues and causing long‐term health damage by mechanism (like increasing the chance for diabetes), PCOS comes with an excessive need for early and precise diagnosis. The correlation between hormone levels and PCOS conditions indicates that endocrine cellular function in epithelial levels has been significantly affected. This opens up the possibility for developing diagnostic biomarkers and miRNAs and small non‐coding RNAs for use in a clinical setting. In this study, we review recent research into how the syndrome exploits miRNAs and lncRNAs in its molecular path and whether this presence and effect makes RNA a biomarker for the diagnosis of PCOS. We also compared the type and methodology of research for prominent groups and their pros and cons. We make the following suggestions for future research: First, there is a need for more meta‐analysis to summarize the statistical data of all the sequencing data from various groups. That is essential in eliminating the errors inherent in sequencing and any bias in selecting the biomolecules of interest in different groups. With rapid sequencing technology, it is not too optimistic to expect reliable RNA biomarkers in clinical use worldwide in the near future. Second, a great deal of research is needed to find more combinatory RNA networks formed of different types of known RNAs in combination with proteins and functional signaling networks. This is where a great insight for PCOS therapeutics is observed. With more unveiling of the molecular mechanism of PCOS, there will be significant core RNAs discovered as the best candidates for RNA immunotherapy development.The authors declare that there is no conflict of interest.Mona Tamaddon: Conceptualization ; Visualization ; Writing – original draft . Mostafa Azimzadeh: Writing – original draft . Seyed Mohammad Tavangar: Conceptualization ; Project administration (lead); Supervision (lead); Writing – review & editing (lead).


Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg , a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the  Positive-sense single-stranded (+ss) RNA viruses are an enormously diverse class of animal, plant and bacterial viruses. Despite this diversity, +ssRNA viruses share a number of key features which facilitate their successful propagation. Notably, the ubiquitous RNA-dependent RNA polymerase is conserved across all +ssRNA viruses, as are its associated functions, targets, main structural architecture and seven highly conserved functional motifs ,2,3. RNAThe attachment of one or more oligonucleotides, such as a GTP (guanylylation) or UTP (uridylylation) to a target protein, can influence the function of the recipient enzyme or protein. Nucleotidylylation of viral proteins by RNA polymerases is therefore a mechanism by which viruses can regulate protein functions during the viral lifecycle. In addition, RNA polymerases implicated in the nucleotidylylation of other viral proteins have also been shown to be nucleotidylylated.Analysis of positive-strand genomic RNA synthesis by a range of +ssRNA viruses shows that initiation can occur via a protein primer-dependent mechanism regulated by the nucleotidylylation of a VPg protein  ,5,6,7. WCaliciviridae and Potyviridae families, and the details of nucleotidylylation in astroviruses and sobemoviruses remain to be elucidated. Viruses within the order Nidovirales also undertake protein nucleotidylylation; however, they do not encode a VPg protein, nor has a direct role for the nucleotidylylated proteins in priming genome replication been described.The biochemical modification of VPg that precedes VPg-mediated priming of viral genome replication, and to a lesser extent the nucleotidylylation of the polymerase itself, has been the basis of a body of research across a number of +ssRNA viral families and is the focus of this review. The picornaviruses represent the most widely studied viral family for nucleotidylylation of VPg proteins, with extensive information for both the biochemical nature of the reaction and the accessory components required for efficient nucleotidylylation. In contrast, less is known about VPg nucleotidylylation in the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae viral families  to prime replication of the viral genome. A tyrosine residue in VPg provides the free hydroxyl for the nucleotidylylation reaction in the lylation ,26,27,28lylation .2+ or Mn2+ [a of the hydroxyl group , making it an effective nucleophile. The hydroxyl group then performs a nucleophilic attack on the alpha phosphate of an NTP. The result is the formation of a phosphodiester bond between the nucleophile and the alpha phosphate and protonation of a pyrophosphate molecule, a by-product of the reaction , although the exact biological role is not known [Nucleotidylylation reactions utilizing asparagine, lysine and possibly serine have also been described for viruses in the e family ,37. Lysiot known ,37.Picornaviridae family comprises a large group of non-enveloped positive-sense single-stranded RNA viruses. The genome is translated using an internal ribosomal entry site (IRES) and produces a large polyprotein that is cleaved to its constituent proteins  does occur, a vast majority of infections are asymptomatic or only cause mild infection [The proteins . For entnfection . The abunfection ,41,42. Anfection .Picornaviridae [The replication of picornaviruses has been reviewed recently , and foraviridae . Both poaviridae ,47, and Picornaviruses have been shown to hijack a host protein, TDP2, to cleave the phosphodiester bond linking VPg to the genomic RNA, with this RNA becoming the template for translation ,49,50. IPicornaviridae encode one VPg protein, with the exception of FMDV, which encodes three, all of which can be uridylylated [Viruses in the ylylated ,59. VPg ylylated   and the 3CD protein (protease and polymerase precursor protein).Precursors of VPg include the 3AB and 3BC proteins, both of which have been proposed as sources of VPg for uridylylation ,63,64. IPoliovirus 3CD lacks polymerase activity but retains protease activity, and interestingly, this protein can stimulate uridylylation by 100-fold in in vitro assays ,75,76. TIn picornaviruses, formation of VPgpUpU is reliant on the CRE, which functions as a template for the uridylylation reaction ,79,80. CCurrently, a “slide-back” mechanism for uridylylation of VPg on the CRE RNA is proposed, which utilizes a conserved 5′AAACA 3′ motif, representing nucleotides five through nine of the CRE RNA loop ,87. For Two binding sites have been described for VPg on the polymerase, leading to two different models for the uridylylation reaction. In the first of these, VPg binds to the ‘front’ of the polymerase and presents the conserved tyrosine for nucleotidylylation into the active site of Pol ,91,92. TIn contrast, uridylylation of enterovirus 71 has been proposed to occur in trans as VPg binds to the polymerase at the base of the palm domain, termed site-311 . SubstitAside from VPg, other viral proteins and protein precursors are able to be uridylylated. In the presence of a 15 nucleotide adenylate template, 3Dpol and 3CD become uridylylated . This ur2+ [2+, no uridylylation is detectable [Uridylylation of 3BC and 3BCD can also be detected in in vitro reactions . Mutatio2+ , but in tectable . WhetherThe overall picture that is emerging for the replication of picornavirus genomes is outlined below. The VPgpUpU formed on the CRE of the positive-sense RNA is transferred through an unknown mechanism to the 3′ end of the poly (A) tail of the positive-sense RNA. The polymerase can then extend the RNA using uridylylated VPg as the primer to produce the negative-sense viral RNA with VPg covalently attached to the 3′ end . HoweverFor synthesis of the positive-sense RNA, the double-stranded replicative form of the RNA must be partially unwound or destabilized to allow uridylylated VPg to transfer to the 3′ end of the negative-sense RNA. As the negative-sense RNA begins with AA bases, uridylylated VPg base pairs with these and the polymerase again elongates the RNA using the negative-sense as a template, thus yielding positive-sense genomic RNA with VPg linked at the 5′ end.Overall, this paints a complex picture of nucleotidylylation in vivo, encompassing both viral and host proteins in the presence of RNA to achieve efficient replication. Dissecting the role of individual components in relation to RNA replication remains an ongoing objective in the understanding of this complex process.Caliciviridae is a family of +ssRNA viruses, comprising 11 viral genera that infect mammals , birds (Bavovirus and Nacovirus genera) and fish  [Norovirus), rabbit hemorrhagic disease virus , and feline calicivirus . genera) , causingNotably, noroviruses became the most common cause of viral gastroenteritis following the global introduction of rotavirus vaccines ,104,105.Calicivirus genomes carry a VPg covalently bound to the 5′ end, whilst the 3′ end has a poly (A) tail. The genome usually encodes three open reading frames (ORF1–3), the first of which encodes all the non-structural proteins, whilst ORF2 and ORF3 encode the major and minor capsid proteins. The ORF1 non-structural gene is translated to a polyprotein which is post-translationally cleaved into precursor and mature proteins by a 3C-like protease (NS6) .Within caliciviruses, the length of VPg proteins can vary from 65 amino acids  to 138 amino acids  . The strNucleotidylylation is an essential step in the viral lifecycle of caliciviruses, with nucleotidylylated VPg acting to prime transcription of the positive-sense genome by viral RdRp in vitro ,113 and Nucleotidylylation of VPg by RNA polymerases, specifically the addition of GTP or UTP , facilitCaliciviridae family that this is more variable. In MNV and HuNV, nucleotidylylation is catalyzed by both forms of polymerase, the mature RdRp and the ORF1-derived precursor protein comprised of unprocessed protease-polymerase  [The biomolecular mechanisms behind nucleotidylylation in caliciviruses are not well understood. In contrast to poliovirus nucleotidylylation, where 3Dpol is the active form of the polymerase responsible for catalyzing the reaction, it has been shown for the rus 3CD) ,121. In rus 3CD) ,123. Prorus 3CD) ,125. In The ability of calicivirus RdRps to nucleotidylylate target proteins from other viruses varies. HuNV RdRp specifically nucleotidylylates only HuNV VPg; in contrast, MNV RdRp is able to nucleotidylylate both HuNV VPg and MNV VPg . Interes2+ as a divalent metal cation, whereas mature polymerase is more active in the presence of an RNA template, regardless of the cation available [The evidence for RNA structures contributing to the nucleotidylylation of calicivirus VPg proteins is not obvious. Noroviruses contain evolutionarily conserved RNA structures, which when disrupted, reduce or completely destroy replication of MNV ,128. Evivailable ,27,129. vailable . FCV Provailable ,123.The HuNV MD145 strain VPg has been shown to be preferentially nucleotidylylated with GTP and UTP over other nucleotides, exhibiting a 2-fold preference for GTP over UTP . A similde novo mechanism [Both UTP and GTP may be added to VPg, and some evidence suggests that two nucleotides can be added to calicivirus VPg proteins , similarechanism . If VPg echanism .To date, the mechanisms behind nucleotide discrimination in nucleotidylylation remain unclear. An NTP-binding sequence has been identified in calicivirus VPgs , suggestPotyviridae viral family and have a wide range of plant hosts, including many vegetables and legume species which constitute important parts of the global food supply. Potyviruses are some of the most common plant viruses in the world, accounting for approximately ~30% of known plant pathogens. Potyviruses are known for causing extensive economic damage to important crops such as potatoes, turnips, tomatoes, beans and peas. Potyviruses are primarily transmitted from plant to plant by arthropods [Potyviridae have a monopartite +ssRNA genome of 8–11 kb in length. Potyvirus genomes encode an ORF which is translated to a 340–370 kDa polyprotein, which is then co- and/or post-translationally processed by three forms of internally encoded protease into cleavage intermediates or mature protein products  RNA template ,141. A cNidovirales encompasses 14 families of positive-sense RNA viruses, within which 109 species have currently been identified [Arteriviridae, Mesoniviridae and Coronaviridae. Arteriviruses are known to infect a diverse group of mammals including pigs , horses  and monkeys (simian hemorrhagic fever virus) [Mesoniviridae family infect mosquitos, and due to the importance of mosquitos in vector transmission, it is an area that warrants further research [Coronaviridae family include toroviruses, which primarily infect livestock, although some strains have been identified to cause disease in humans [The order entified . Of thisr virus) . PRRSV ir virus) . Virusesresearch . Coronavresearch . Generalresearch . Other vn humans ,153.The RdRps of coronaviruses  are made up of three main domains: the RdRp domain, the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain and an uncharacterized third domain that connects the NiRAN to the RdRp domain . In vitrWhile coronaviruses do not possess a VPg protein as described for other viruses in this review, they perform nucleotidylylation as a critical component of the viral replication cycle. Currently, there is only experimental data on nidovirus nucleotidylylation from four viruses: EAV, HCoV-229E, SARS and SARS-CoV-2. As with the other ‘branch 2′/picornavirus supergroup of viruses  describeThe preference of the incorporated nucleotide during nucleotidylylation by SARS-CoV-2 RdRp and HCoV-229E RdRp is UTP over GTP with the HCoV-229E activity being 2–3-fold higher using UTP . HoweverCoronaviridae, Toroviridae, Mesonivirdae and Arterivirdae families [n, Bn and Cn (Nidovirales, with no homologs identified elsewhere [Analysis of nidovirus polymerase sequences has identified a unique domain in the N-terminal portion of nidovirus RdRp, termed a NiRAN domain. Alignments of this domain between viruses from families  showed sn and Cn . Mutatiolsewhere ,157. WheArteriviridae family [Each of the RdRps described above were identified to nucleotidylylate other non-structural proteins. The nucleotidylylation activity of EAV RdRp was notably enhanced when in the presence of nsp7 . Nsp7 ise family  and is pe family . The role family . Three se family . The rolThe nsp9 of both HCoV-229E and SARS-CoV-2 are nucleotidylylated by their respective RdRps. Both viruses preferentially use UTP as a substrate; however, GTP can be utilized ,156. NspWhile HCoV-229E RdRp was not seen to nucleotidylylate nsp7 in an in vitro assay , the SARNucleotidylylation activity is essential for coronavirus replication, with nucleotidylylation knockout mutations resulting in a nonviable virus. Slanina et al.  proposedYan et al. showed that the NiRAN domain of nsp12 is able to catalyze the transfer of GMP to ppA, which forms the GpppA cap core structure. They suggested that the association of the nsp9 to the NiRAN domain may stabilize the 5′ end of GpppA-RNA due to nsp9 being an RNA-binding protein before the GpppA-RNA is further processed during cap synthesis .Protein nucleotidylylation is a critical component of the replication of a range of viruses, with this modification commonly found in +ssRNA viruses of the picornavirus supergroup , and hig