Systematics is described for annotation of variations in RNA molecules. The conceptual framework is part of Variation Ontology (VariO) and facilitates depiction of types of variations, their functional and structural effects and other consequences in any RNA molecule in any organism. There are more than 150 RNA related VariO terms in seven levels, which can be further combined to generate even more complicated and detailed annotations. The terms are described together with examples, usually for variations and effects in human and in diseases. RNA variation type has two subcategories: variation classification and origin with subterms. Altogether six terms are available for function description. Several terms are available for affected RNA properties. The ontology contains also terms for structural description for affected RNA type, post-transcriptional RNA modifications, secondary and tertiary structure effects and RNA sugar variations. Together with the DNA and protein concepts and annotations, RNA terms allow comprehensive description of variations of genetic and non-genetic origin at all possible levels. The VariO annotations are readable both for humans and computer programs for advanced data integration and mining. RNA variations are increasingly in the focus of molecular biology, genetics and medicine. Known disease-causing RNA variants mainly affect proteins. Non-coding RNAs have gained interest and have been shown to have numerous functions. Nowadays, tens of types of RNA molecules are known. Those are functional or related to numerous cellular processes and functions ranging from catalysis to regulatory processes, from information transfer for protein synthesis to function in cellular machineries, from RNA base and sugar modification to RNA interactions, and so on.http://variationontology.org/) was developed for systematic description of variations and their consequences, effects and mechanisms [Terminology for RNA molecules, functions and processes is still confusing. There is not even consensus for the definition of RNA – apart from being chemically a polynucleotide . Common chanisms . VariO iVariO annotations can be made on three molecular levels – DNA, RNA and protein – and for four types of information: variation type, structure, function, and properties. This article concentrates on RNA variations, detailed descriptions of DNA and protein variants have been published previously ,4. VariOWhen using VariO, systematic annotations, include the VariO prefix and a number followed by the term. The notation VariO:0319 means the same as the name of the term, i.e. ‘RNA deletion’. When using annotations, both the number and the prefix ‘VariO’ must be included. Although the names of the terms can be obtained automatically, full names must be provided when the information is intended to be read by people. This article follows the VariO RNA term hierarchy on the four sublevels . The headings and subheadings are VariO terms. For clarity, VariO terms are written in quotation marks in the text. We have published guidelines for the annotation process . The varhttp://variationontology.org/) including Ontology Lookup Service (https://www.ebi.ac.uk/ols/ontologies/vario), OBO Foundry (http://www.obofoundry.org/ontology/vario.html), NCBO BioPortal  Ontobee (http://www.ontobee.org/ontology/VariO), FAIRsharing (https://fairsharing.org/bsg-s000776/) and others.VariO is available in several ways in addition to the ontology website . In DASHR  there arLncVar contains variations in long non-coding RNAs (lncRNAs) . lncRNAsT-psi-C is a database for tRNA sequences and structures . InformaIn addition to splicing, RNAs undergo various other modifications, data for modifications, pathways, modifying enzymes and modification locations within sequences are available in MODOMICS . siRNAmoSubcellular localization of RNA molecules varies, details are in RNALocate . RNAs haRNA families and structures are classified in several levels in RNArchitecture . BGSU RNSeveral resources have been developed for RNA data in relation to cancers. YM500v3 . Lnc2CanThe first category is ‘VariO:0306 RNA variation type’, which provides a brief description of the variation with commonly used terms . These tBTK [BTK coding region [There are eight categories of RNA chain variation types, some of them with subcategories . r.3g>u BTK  can be aBTK . The senBTK . In ‘Varg region . ‘VariO:NPC1 coding for NPC intracellular cholesterol transporter 1 causes Nieman-Pick type C disease due to ‘VariO:0245 missing RNA’ [NPC1 variants with premature stops.RNA quality control mechanisms, like nonsense-mediated decay (NMD) and staufen-mediated decay (SMD), degrade mRNA molecules that contain premature stop codons. Homozygous c.2978delG variation in ing RNA’ . NMD redBTK gene causes ‘VariO:0319 RNA deletion’ r.777_839del [BTK gene leads to p.L652* protein truncation and XLA [Substitution c.839+5G>A in 7_839del ,64 and s and XLA .BTK [BTK [‘VariO:0311 RNA indel’ contains both inserted and deleted components. An example is r.1682_1683delinsa in BTK  which isBTK [BTK  is a ‘VaBTK r.1812_1813insgacagu is a ‘VariO:0326 RNA insertion’ introducing additional nucleotides. This XLA-causing variant [ variant  is of tyKISS1R for KISS1 receptor 1 modifies stop codon and causes normosmic congenital hypogonadotropic hypogonadism [When a sequence stretch in RNA is moved to a new location it is called for ‘VariO:0241 RNA translocation’. In ‘VariO:0244 RNA inversion’ sequence is inverted to its original place. If the stop codon is altered to code for an amino acid ‘VariO:0309 termination codon change’ occurs. r.1195u>c transition in gonadism .BTK r.1559g>a missense variation leading to p.P520Q substitution [RNA variation origin has two subclasses, ‘VariO:0307 RNA variation of genetic origin’ and ‘VariO:0333 RNA variation of non-genetic origin’ . Varianttitution  is of geADA) gene has nine alternative transcripts which are of type ‘VariO:0329 alternative RNA form’, see LRG_16 in Locus Reference Genomic database [BTK construct is a ‘VariO:0247 artificial RNA variation’ [NPC1 is recognized by NMD [Several variation types are of non-genetic origin. Adenosine deaminase  gag-pol fusion protein  is due tgag-pol is also an example of ‘VariO:0408 RNA chimera’. Chimeras can emerge with different mechanisms including, e.g., read-throughs of adjacent genes, juxtaposition of transcripts encoded by genes on different chromosomes, and from noncontiguous genes within the same chromosome. Human yippee like 5 (YPEL5) and protein phosphatase 1 catalytic subunit beta (PPP1CBI) genes form recurrent and reciprocal chimeras in chronic lymphocytic leukaemia [HIV-1 eukaemia .MIR140 leads to gain of function activity and causes human skeletal dysplasia [‘VariO:0396 variation affecting RNA function’ has six categories. Mitochondrial m.1616A>G substitution in tRNA-Val gene and its RNA form r.1616a>g cause mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome . It is aysplasia  due to ‘BTK [BTK causes XLA because of deletion of residues 260–280 from the protein distorts the structure of the Src homology 3 (SH3) domain [r.3g>u substitution in BTK  initiati) domain . This isBTK has ‘VariO:0304 association of RNA variation to pathogenicity’ [NPC1 mRNA is degraded by NMD and thus has ‘VariO:0010 effect on RNA abundance’ [Variations affect several properties of RNAs and have general annotation ‘VariO:0298 variation affecting RNA property’. XLA-causing r.1559g>a missense variation in enicity’ . It has undance’ .Tetrahymena thermophila group I ribozyme to optimize and to have ‘VariO:0442 effect on RNA specificity’ [RNA catalytic activity changes are described with ‘VariO:0439 effect on RNA activity’ and terms at three more specific terms. Replacements at conserved adenosines 248 or 249 in the J5/15 region of RNase P, a ribozyme, have ‘VariO:0441 effect on RNA affinity’ . Variantificity’ . These vMT-TV leads to very low steady-state levels (<1%) of normal mt-tRNA-Val because the variant tRNA remains deacylated and is rapidly degraded [BEAN1) and thymidylate kinase 2 (TK2) are responsible for spinocerebellar ataxia type 31 (SCA31) [Degradation of RNA molecules is important for control of their function. Most RNA molecules are short-lived. m.1625c>t substitution in mitochondrial tRNA-Val gene degraded . Individ (SCA31) . These RFTL) gene cause hereditary hyperferritinemia – cataract syndrome (HHCS) with increased serum ferritin levels and early-onset cataracts [Iron-responsive elements (IREs) are RNA interaction motifs. Variations at IRE in the transcripts for ferritin light chain (ataracts . IRE motDMPK) transcripts form labile aggregates and are annotated with ‘VariO:0364 effect on RNA aggregation’ [CNG-triplet repeats (N indicating any nucleotide) are frequently behind neuromuscular diseases. CUG-repeats in DM1 protein kinase (egation’ .DIAPH) mRNA localizes to endoplasmic reticulum in fibroblasts independent of zipcodes. Frameshift-causing variant in DIAPH loses perinuclear localization of the transcript [FTL double variant r.18c>u, r.22u>g (Pavia2) has ‘VariO:0301 effect on RNA stability’ since it reduces the thermal stability of the IRE-containing RNA.Many mRNAs that are directed to compartments contain one or multiple localization signal sequences (zipcodes), which are recognized by zipcode binding proteins. Diaphanous-related formin 1  is related to hypertension because of ‘VariO:0308 variation affecting RNA structure’ [r.22u>c transition at the D-stem in mitochondrial ructure’ .Genomes in many organisms are pervasively transcribed to large spectrum of RNA forms. There are two major types of affected RNA type, namely ‘VariO:0350 non-protein coding RNA’ and ‘VariO:0351 protein coding RNA’, see Classification principles have been presented for long non-coding RNAs ,88, howeDMPK) gene contains CTG repeats. Healthy people have few tens of repeats while patients with myotonic dystrophy type 1 (DM1) have more than 100 repeats, often even thousands of them. The repeats are part of CTCF insulator that regulates the expression of DMPK. An antisense transcript extends from the regulatory region of the adjacent gene to the CTF repeats. Extensive number of repeats affects the length of the antisense transcript and thereby gene regulation [Antisense RNA molecules are complementary to mRNA region, while sense RNA overlaps with mRNA, but is not involved in protein coding. Both are single-stranded molecules. ‘VariO:0463 Antisense RNA’ can block translation by hybridizing with mRNA. These transcripts are important regulators of protein expression and have biotechnological and therapeutic applications. The 3ʹ UTR region of DM1 protein kinase  proteins [The regulation of nse RNA’ . Congeniproteins . The pro‘VariO:0353 non-coding region of mRNA’ in the mature mRNA contains the 3ʹ and 5ʹ flanking regions, which are essential, e.g., for regulation and translation.‘VariO:0465 Functional non-coding RNA’ has several subclasses . SeveralHBB) gene [Genome editing, most notably with systems developed from the bacterial antiviral defence system with CRISPR/Cas9, is now widely used in research laboratories and biotechnological applications are under development. Editing is facilitated by guide RNA, a form of ‘VariO:0484 DNA editing-related RNA’ that directs the Cas9 nuclease to digest and remove or add new genetic material. The guide RNA detects a specific DNA location to be edited based on sequence complementarity. One of the early examples is the correction of a β-thalassaemia-causing double variant in human haemoglobin subunit beta (BB) gene . This vaSNORD50A) gene appears frequently in prostate [SNORD50A expression is reduced due to the variation. The expression of wild-type form inhibits cancer cell growth.mRNA editing in humans and many other organisms is not RNA guided. Small nucleolar RNAs (snoRNAs) in many organisms and guide RNA (gRNA) in kinetoplastid protists are involved in RNA editing as ‘VariO:0483 RNA editing-related RNA’. snoRNAs guide methylation and pseudouridylation especially of rRNAs. Deletion of two bp (TT) from the small nucleolar RNA, C/D box 50A  pathway to regulate gene expression by directing targeted mRNAs for degradation and thus preventing protein production. Although siRNAs can be designed to silence disease-related variants, it is a daunting task where various aspects and predictions have to be taken into account . This ar‘VariO:0480 Piwi-interacting RNA’, piRNA, interacts with piwi-subfamily Argonaute proteins that are mainly involved in post-transcriptional silencing of transcripts for repeat sequences, especially of transposable elements.RNU4ATAC gene for RNA, U4atac small nuclear (U12-dependent splicing) caused microcephalic osteodysplastic primordial dwarfism type I (MOPD I) with associated pigmentary disorder [mRNA is spliced in a large protein-RNA complex that contains ‘VariO:0485 spliceosomal RNA’. Homozygous g.55G>A variation in disorder . The encXist is a lncRNA and ‘VariO:0482 X-chromosome inactivation center RNA’ functional in X-chromosome inactivation (XCI) where one copy of the X-chromosomal genes is inactivated in females to compensate for the presence of only one copy in males. Deletion of one of the highly conserved Xist-specific repeat elements, repeat E, increases the expression of a number of XCI escape genes [pe genes .Telomeres are repetitive regions in the termini of chromosomes. They protect the chromosomes and are shortened gradually during chromosome replication as the Okazaki fragment binding region is not copied. Telomerase is a ribonucleoprotein complex that adds telomere repeats to the 3ʹ ends of telomeres to compensate for loss of sequence during replication. ‘VariO:0475 telomerase RNA’ is used as the template for repeats. Variations in telomerase RNA are associated for example to dyskeratosis congenita and aplastic anaemia. Cellular experiments show that disease-associated point variants in the pseudoknot and template regions of telomerase RNA lead to lower level of the RNA and much shorter telomeres .Several RNA components act as ‘VariO:0468 translation-related non-coding RNA’. Proteins are synthesized at ribosomes that consist in addition to several protein components also ‘VariO:0493 ribosomal RNA’ in the subunits that are annotated as ‘VariO:0469 large subunit ribosomal RNA’ and ‘VariO:0470 small subunit ribosomal RNA’. m.1555A>G substitution in the small mitochondrial subunit 12S rRNA is associated with non-syndromic deafness . The patBTK intron 3 position −1 causes exon skipping and is a spliceosomal intron variation [‘VariO:0471 intron’ is a non-coding region located between exons in a pre-mRNA and are cleaved during maturation. Introns appear also in many non-protein coding RNAs from which they are cleaved during maturation. ‘VariO:0473 spliceosomal intron’ is cleaved at spliceosome. tRNA intron is cleaved by a tRNA splicing endonuclease, while ‘VariO:0472 self-splicing intron’ is removed autocatalytically. r.777_839del in ariation . ‘VariO:ariation .Some 600 tRNA genes are coded by the human genome. Human mitochondrial genome codes for 22 tRNAs, the other tRNAs are imported nuclear-origin molecules. Several diseases are caused by variants in mitochondrial tRNA genes. PON-mt-tRNA is a predictor for disease relevance of mitochondrial tRNA variants . PredictCertain RNA molecules are located to special compartments or cellular regions, there are also nuclear and mitochondrial RNAs, described by ‘VariO:0486 location or origin of non-coding RNA’. DM1-causing CTG repeats in the antisense RNA molecule  are in ‘‘VariO:0491 size of non-coding RNA’ can be classified into three categories. ‘VariO:0492 short non-coding RNA’ molecules are shorter than 200 nucleotides, often substantially shorter than the threshold. miR-140 which contains variants in skeletal dysplasia miRNA  is a shoTTN for titin contains 363 exons [There are several forms also of ‘VariO:0351 protein-coding RNA’. Alternative splicing is a common mechanism regulating gene expression  and incr63 exons . The shoRBFOX1 of 2,473,592 bp, the longest mRNA is for TTN, 109,224 bp. Variations in ‘VariO:0352 coding region of mRNA’ lead to many types of protein variants, discussed in [‘VariO:0460 pre-mRNA’ is matured to ‘VariO:0461 mRNA’ via several processing steps. pre-mRNAs constitute substantial part of heterogeneous nuclear RNA (hnRNA) and contain many very long RNA molecules. The longest human gene is for RNA-binding fox-1 homolog 1 One of the mRNA maturation steps is excision of introns during splicing. Alternative splicing is common, some 95% of multiexon genes could undergo alternative splicing , but it ALK) is frequent in melanomas and appears also in some other cancer types [Although alternative splicing produces large numbers of variant proteins, alternative start and termination produce even wider range of variation . They deer types . The novAddition of polyadenylation signals to 3ʹ end is one of mRNA maturation processes. Heterogeneity can appear at the polyadenylation tails and this can lead even to diseases as the poly-A tails are targets for miRNA regulation. ‘VariO:0356 alternatively polyadenylated mRNA’ due to single nucleotide variations can affect transcript length and gene expression . Variati‘VariO:0462 circular RNA’ is a covalently closed single-stranded RNA ring. circRNAs are formed via splicing and can code for proteins. These molecules are formed when an upstream splicing acceptor joins with a downstream splice donor by back-splicing mechanism. circRNAs are common and have roles both in diseases and development . hsa_cirAlthough DNA is the most common polynucleotide for genomic information in nature, ‘VariO:0456 RNA genome’ contains the genetic material in many viruses. ‘VariO:0457 RNA virus genome’ is usually single-stranded. According to the International Committee on Taxonomy of Viruses (ICTV) RNA viruses are classified to Group III, Group IV or Group V in the Baltimore classification system . As RNA Coleus blumei viroid 1 (CbVd-1) variants clone 1 (accession number MG767212) and clone B (DQ178395) differ at position 25 and have different seed-transmission frequencies, 30% to 0% [Satellite panicum mosaic virus coat protein Kansas isolate (SPMV-KS) affect interaction with the helper Panicum mosaic virus [Viroids are the smallest known pathogens, they appear in plants. ‘VariO:0458 viroid genome’ contains just a single-stranded, circular RNA without any protein coating or other molecules. Viroids are classified as subviral agents by ICTV. 0% to 0% . Satelliic virus .Effect on post-transcriptional RNA modification includes variations that can be described either with ‘VariO:0498 effect on RNA modification’ or ‘VariO:0362 effect on RNA splicing’, see MT-TE replaces highly conserved uridine r.55u>c in TΨC loop that is modified to pseudouridine and affects the conformation and stability of the tRNA molecule leading to maternally inherited diabetes and deafness [Both noncoding, including tRNAs, rRNAs, spliceosomal small RNAs, etc., and coding RNAs (mRNAs) frequently contain nucleotide modifications. Collectively the different RNA forms are known to have more than 100 distinct modifications, see MODOMICS database . m.14692deafness . The varDMD) is the largest human gene, it contains 79 exons. Some of the introns are subject to non-sequential and recursive splicing [COL1A1) causes osteogenesis imperfecta due to splicing defect [In addition to mRNAs, e.g., tRNAs, rRNAs, lncRNAs, ribozymes and circRNAs contain introns or spacers that are removed during maturation before ligating the ends of the chains. Variations at canonical and noncanonical splice sites and those introducing cryptic splice sites can alter mRNA structure and have ‘VariO:0362 effect on RNA splicing’. The ‘VariO:0509 effect on mode of splicing’ has three subterms that describe the type of splicing process affected . Splicinsplicing , where vg defect . The varGLA gene for galactosidase alpha leads to two aberrant transcripts [VWF) gene causes type 1 von Willebrand disease [nscripts . In one, disease . Intron SERPING1) gene creates a donor splice site in intron 6 and causes ‘VariO:0504 inclusion of cryptic exon’ leading to hereditary angioedema (HAE) type I [c.1029+384A>G transition to human serpin family G member 1  type I . This vaMAPT gene affects splicing of mutually exclusive exons and causes frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) [‘VariO:0513 mutually exclusive exon’ annotates situations where only one of two exons is included into mRNA. C>G substitution at position +19 in exon 10 of microtubule-associated protein tau FTDP-17) . MutuallFTDP-17) .RPS6KA3 causes partial exon skipping ‘VariO:0502 loss of exon fragment’ and leads to a premature termination codon [Variants in ribosomal protein S6 kinase A3 (RPS6KA3) cause Coffin-Lowry syndrome (CLS) with variable phenotypes, e.g., with digital and facial anomalies as well as syndromic intellectual disability. c.613G>C in on codon . The patBTK gene causes XLA due to ‘VariO:0502 exon skipping’ and deletion of 21 residues [BTK causing r.392_520del [Substitution c.839+5G>A in residues . The del2_520del  is a ‘Va2_520del . AlteratAll the examples above are of type ‘VariO:0365 effect on cis splicing’. ‘VariO:0376 effect on trans splicing’ is used to describe splicing and ligation of two mRNA molecules producing a chimeric molecule. Trans splicing is actively investigated as a gene therapy modality to correct errors in mRNAs by generating RNA chimeras .Lots of computer predictors have been released for various aspects of splicing. Tools for the effects of variants on splicing, including acceptor and donor splice sites, exonic sequences, exonic and intronic splicing silencers and enhancers, branch point sequences and polypyrimidine tracts were reviewed in . There aN6-methyladenosine (m6A) [5C) [N1-methyladenosine (m1A) and m6A modifications have been benchmarked [Several methods have been developed for RNA modification site , usuallyne (m6A) , 5-methy6A) [5C) , pseudou6A) [5C) ,151 and chmarked . tRNAmodchmarked .The primary RNA structure (sequence) forms secondary structural elements that are central components of the three-dimensional tertiary structure. The folded chain can then form quaternary structures together with RNA and other molecules. There are six types of RNA secondary structures . Stem isMAPT gene exon modifies the structure of stem in the mRNA and increases its stability [Variations in the secondary structural elements have various effects. C>G substitution in tability . FTDP-17tability .Saccharomyces cerevisiae ribosomal protein L30 represses its own splicing and translation. Single and multiple variations affect both protein affinity and repression [L30 transcript tolerate alterations, while changes at sites 10, 11, 12, 58 or 59 have marked effect on binding and regulation [Some variations within hairpin loops cause conformational alterations  and havepression  having ‘gulation .Telomerase complex maintains chromosome telomere length and stability. Telomerase RNA component has a highly conserved pseudoknot, variations in which disrupt the structure and abolish telomerase activity due to ‘VariO:0500 effect on RNA pseudoknot’ . Even ‘VMAPT gene [RNA molecules fold into three-dimensional and quaternary structures and form a large number of structural forms . RNA douAPT gene  is an exAPT gene . These rAPT gene  . These changes are not inherited and are thus not epigenetic. Gene expression regulation, e.g., by non-coding RNAs is not an epigenetic trait either, it is one form of regulation. These changes are annotated with ‘VariO:0354 effect on post-transcriptional RNA modification’ and ‘VariO:0498 effect on RNA modification’, similar to post-translational modifications in proteins. There are a few verified examples where short RNA molecules are involved in epigenetics, however even these are not ‘VariO:0438 epigenetic RNA modification’, as the epigenetic effect is not on RNA level.RNA related research is advancing at long paces. New RNA forms are reported frequently and novel insights are obtained on the function and importance of the various transcripts. Thus, there is immediate need for systematic description of RNA related information to facilitate data mining, integration and analysis also from several sources. Comprehensive conceptualization of RNA variations was implemented into VariO to facilitate detailed description of all kinds of RNA variants, effects, consequences and mechanisms. Consistent annotations can be made with VariOtator tool . RNA ter


Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x–7.5x), with a consistently observed favoured 5’ U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11–14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including  The rapid evolution of RNA viruses is thought to arise from high mutation frequencies during replication and the rapid accumulation of genetic changes over time in response to its changing environments. This study describes an additional potent factor that contributes to the evolution of RNA viruses infecting mammals, the occurrence of specific C->U mutations in a subset that possess intensely structured genomes. This mutational process substantially damages the virus’s ability to replicate and is potentially akin to “genome editing” of HIV-1 and other retrovirus genome sequences by APOBEC, one of the principal components of vertebrate antiretroviral defence mechanisms. This study however provides evidence for a wider mutational activity against several human and veterinary RNA viruses, including HCV and foot and mouth disease virus. The C->U mutational process accounted for 15–20% of standing sequence variability of these RNA viruses, representing a potent driver of their sequence diversification and longer-term evolution. The evolution of viruses is typically conceptualised as a combination of adaptive sequence change in response to a range of selection pressures in the environment and a process of random diversification in which neutral or near neutral nucleotide substitutions become fixed in virus populations –3. An exA further source of mutations in viruses arises from effects of several innate antiviral effector mechanisms in vertebrate cells that operate through viral genome editing. Of these, the best characterised are the interferon-inducible isoform of adenosine deaminase acting on RNA type 1 (ADAR1) that tarThe current study investigated this possibility through construction and analysis of sequences changes in alignments of mammalian RNA virus sequences from a wide range of families. Since APOBEC-mediated RNA editing has been proposed to occur in the context of RNA structure elements, virus datasets were additionally scanned for sequence-dependent internal RNA base-pairing ,20. ViruThe primary resource for the study were aligned sequences from a wide range of mammalian RNA viruses derived from several vertebrate virus families Tables  and S1. The previous analysis of the directionality of sequence changes in SARS-CoV-2 and the detection of an excess number of C->U changes was simplified by the minimal sequence diversity of the assembled post-pandemic sequences . SARS-CoSequence datasets was analysed using the program Sequence Change in the SSE package, which records the occurrences and sites of sequence changes from a majority rule alignment consensus sequence. Collectively, there was a significantly greater number of C->U changes compared to its reverse (U->C) and to other transitions in the RNA virus datasets at sites showing <5% heterogeneity although the degree of over-representation was highly variable between viruses . TransitTo more formally quantify the degree of over-representation of C->U transitions in each virus dataset, the ratio of each transition to its reverse . Similarly for HCV-1a, both U->C and C->U transition frequencies followed marginally skewed Poisson distributions (test statistics 1.5 [p = 0.03] and 2.5 [p < 0.001] respectively), but with a higher mean number of C->U transitions per sequence (12.1 / sequence) than the reverse (2.8 / sequence). The sequence datasets of HCV and EV-A71 sequences therefore showed no evidence for the occurrence of individual hypermutated sequences as described previously for HIV-1 [The estimation of relative transition frequencies was collectively based upon all sequences within each virus alignments. To investigate the degree of heterogeneity in C->U changes between sequences, numbers of this transition were computed individually and compared with those of the reverse mutation  in two oor HIV-1 –25; the p = 0.002). There was no association with MFE, representing the minimum free energy on RNA folding, a property primarily influenced by the G+C content of the sequence, which also showed no association with the C->U / U->C transition asymmetry. There were similarly no associations with extents of CpG or UpA dinucleotide suppression in RNA virus genomes, or base imbalances . As expected from the minimal differences from the null expectation, no association between G->A/A->G transition asymmetry values with any compositional or structural sequence attribute was detected.To investigate which genome features of RNA genomes were predictive of the C->U/U->C transition asymmetry, a range of compositional attributes , mean folding energies (MFEs) of consecutive 300 base fragments and differences of this value from sequence order randomised controls (MFEDs) were computed. The association of each with C->U/U->C and G->A/A->G transition asymmetry values was analysed by multivariate analysis . MFED vap values of 0.0004, 5 x 10−11 and 8 x 10−5 respectively by Mann Whitney U test). Effects of 3’ context were far more variable between viruses, but with evidence for favoured C->U transitions upstream of A in FMDV and to lesser extents in other RNA viruses.C->U transitions in SARS-CoV-2 genomes were influenced by the immediate 5’ and 3’ base contexts of the mutated site . Other RPrevious analyses demonstrated that a proportion of C->U mutations in SARS-CoV-2 failed to become genetically fixed in a population . The disThe program, Homoplasy Scan in the SSE package was developed to sequentially analyse each variable site in a virus sequence alignment; this recorded the degree of segregation of each base in a global tree constructed from 1200 base genome fragment that incorporates the interrogated bases . AssociaBroadly, the observed differences arose from different genetic structuring of sample populations; phylogenetic trees from alignments with predominantly informative sites showed a marked degree of structured internal branching , while vIrrespective of the actual distributions of AI values (and associated differences in tree topologies of the different RNA virus datasets), categorisation of sites based on AI value ranges allowed an investigation of whether C->U transition frequencies were specifically over-represented at phylogenetically uninformative sites as would be expected if these mutation were subject to homoplasy . AmongstThe extent to which the observed excess of potentially homoplastic and transient C->U mutations in certain viral datasets contributed to overall viral sequence diversity was calculated. The number of sites in a virus alignment showing a majority C->U change subtracted by those showing majority U->C changes (excess C->U) were expressed as proportion of the number of variable sites in the alignment, with totals sub-divided into sites showing different ranges of AI values . There wThis study provides evidence for an excess of C->U changes over the reverse (U->C) and complementary (G->A) transitions in a diverse range of RNA viruses, including HCV , FMDV, MNV, HPgV-1 and rubella virus. Fold excesses ranging from 1.9x – 3.6x  approachThe underlying mechanism(s) for the observed elevated frequencies of C->U changes in certain RNA viruses are functionally uncharacterised and conceivably may originate through several mechanisms. In the discussion, we will briefly review the evidence for or against transcriptional, RNA damage-associated and cellular RNA editing mechanisms that may create the excess C->U mutations observed in RNA viruses, taking into account their prominent (+)strand asymmetry, 5’ base context effect and their variable distributions in viruses with structured and unstructured genomes.A major source of mutations in viruses and other organisms are transcription errors by cellular RNA or DNA polymerases that are incorporated during replication. Different types of polymerases may show varied mutational profiles with separate propensities to mis-incorporate particular transitions or transversions. Very limited data exists on mutation frequencies associated with viral RNA dependent RNA polymerases (RdRps). However, biochemical assays of misincorporation kinetics on RNA templates by the poliovirus RdRp ,33 or reMutations introduced by RNA transcription would be expected to be symmetric in RNA viruses as their genomes derive from an equal number of positive and negative strand copyings by the same RdRp operating in the same cellular compartment. A tendency to misincorporate a U instead of a C would therefore be reflected in a parallel number of G->A mutations where it occurred on the minus strand. However, as observed previously for SARS-CoV-2 –14,26, tTo counter this, it could be argued that natural selection operates differently on the minus- and plus-RNA strands where the limited number of minus-strand templates produces a much larger number of genomic RNAs. Therefore there may be more stringent selection against mutations occurring in the negative-strand as these are invariably copied into the plus-strand, whereas plus-strands are relatively dispensable and mutations occurring during their synthesis will not overly affect the overall replication process. Indeed, full-length transcripts with plus-strand synthesis errors could be readily packaged into virions assembled from viral proteins synthesised from independently transcribed mRNAs.An alternative possibility is that mutations occurring during transcription of the minus strand are more rather than less likely to be fixed because minus strands are in a substantial minority of viral RNA sequences in the infected cell. Mutations arising from specific RdRp misincorporation biases occurring early in infection during synthesis of the minus-strand might be more likely to become fixed in the infected cell through a founder effect than mutations occurring from the 10s or 100s of plus-strands synthesised from that template. In contrast to the selection-based asymmetry hypothesis above, the observed excess of C->U mutations in the genomic (plus-)strand must therefore have originated from elevated frequencies of G->A mutations in the minus-strand rather than C->U.Neither hypothesis for a transcriptional origin of the C->U strand asymmetry recorded in the current study is supported by the observation of its variable presence in different RNA virus groups and association with genomic RNA secondary structure. While it is possible that RdRps from different RNA viruses may vary in their spectra of misincorporation frequencies, it is notable that C->U asymmetries cut across family divisions, with examples of picornaviruses and flaviviruses showing examples of viruses with both biased  and unbiased  C->U transition frequencies. The association of C->U asymmetry with the presence of genomic RNA secondary structure  thereforIt has been recently proposed that substitutions occurring in the SARS-CoV-2 genome, notably the G->U transversion may originate from mutational damage to viral RNA during periods of oxidative stress ,16. ROSsAn alternative source of mutations arises from the documented effects of several innate antiviral effector mechanisms in vertebrate cells that operate through a process of genome editing; these may potentially introduce mutations into RNA virus genomes during replication. Of these, the best characterised are the interferon-inducible isoform of adenosine deaminase acting on RNA type 1 (ADAR1) that tarMembers of the APOBEC family typically target single stranded DNA templates for mutagenesis of cytidine to thymidine during reverse transcription of retroviruses and hepatitis B virus (HBV) and in the genomes of small DNA viruses such as papillomaviruses ,38,39. FIf APOBEC was responsible for the over-representation of C->U changes in RNA virus genomes, there should be an increased C->U/U->C mutational asymmetry downstream of a U, as this is the target preference of most mammalian APOBEC paralogues. Consistently with findings of cellular mRNA editing by A3A ,42 and peg. the relative representation of UpC, ApC, GpC and CpC) to normalise for 5’ compositional effects. However, the problem with this approach is determining 3’ contexts since the CpG dinucleotide is substantially suppressed in mammalian +strand viruses, often to 15–20% of expected frequencies [The wide range of G+C contents of the viruses analysed in the current study  necessitquencies –46; atteThe favoured 5’ context of APOBEC-mediated mutations and consequent depletion of TpC/UpC dinucleotides has been exploited as a means to identify editing “footprints” in viral genomes in a previous bioinformatic analysis . DepletiApart from 5’ and 3’ base contexts, the only other compositional metric influencing the extent of C->U / U->C transition asymmetry was RNA structure formation , a genomThis association with structured RNA virus genomes potentially recapitulates the previous noted restriction of editing of human mRNA sequences by A3A to sites in defined stem-loop contexts . In an aWhile these associations are intriguing, the evidence is circumstantial without systematic functional studies to support a role of APOBEC-mediated RNA editing in restriction of RNA virus replication. Indeed, it was shown that antiviral effects of APOBEC occurred in the absence of any evidence for RNA editing of coronavirus HCoV-NL63 genomic sequences . It woulin vitro and a targeted gene deletion approach to functionally test editing abilities of individual APOBEC proteins on RNA templates would be important steps in such investigations.Inferences on mechanism based on bioinformatics analyses therefore must at this stage be indirect; they should additionally acknowledge that the currently recognised repertoire of RNA editing pathways and other mutational mechanisms in mammalian and other vertebrate cells is almost certainly incomplete. Viral RNA editing may indeed originate from an entirely different mechanism outside the current ADAR1 and APOBEC paradigm. As recently suggested, overlapping but nevertheless distinct C->U mutational contexts in SARS-CoV-2 and rubella genomes points towards the operation of more than one mutational mechanism ; the hetIrrespective of the underlying mutational mechanisms, the analyses performed in the study provided evidence that a substantial proportion of population variability in HCV and other structured viruses could be attributed to a marked over-representation of C->U transitions  at an individual sequence level approximated to a Poisson distribution , albeit While tangential to the primary focus of the study, the shape of phylogenetic trees constructed from different RNA viruses differed substantially Figs  and S2 ahttp://www.virus-evolution.org/Downloads/Software/) [ie. picornaviruses, flaviviruses) was based on coding regions only. Regions spanning the start of the first open reading frame (ORF) to the end of the last ORF were used for analysis of viruses with polycistronic genes . Alignments are available from the author on request.Alignments of sequences of HCV genotypes 1a, 1b, 2a and 3a, SARS-CoV-2 and other coronaviruses were derived from previous studies ,50. Furtftware/) . AnalysiCalculation of pairwise distances and nucleotide composition was performed using the SSE package version 1.4. Sequence changes were compiled using the program Sequence Changes with a variability threshold typically set at 5% heterogeneity, where heterogeneity was calculated as the cumulative frequency of all non-consensus bases. Multiple thresholds were used to analyse mutation representation at sites showing different levels of variability Figs .Normalised ratios (nC-U) of C->U and U->C transitions (and comparably for G->A and A->G) were calculated as:Normalised 5’ and 3’ context preferences for C->U/U->C transition asymmetries (nC->U) in specific 5’ and 3’ contexts, (5’x)nC->U and (3’x)nC->U  were calculated as:Computation of MFE and MFED values was carried out using the program Folding energy scan in the SSE package using sequential 300 base sequence fragments incrementing by 30 bases between fragments. The program call the RNAFold.exe program in the RNAFold package, version 2.4.2  with defet al.[et al. [AI values were calculated using the algorithm originally described by Wang et al. and Coch.[et al.  and implNeighbour joining trees were constructed from aligned sequences using the program MEGA7 . LineageAll statistical calculations and histogram constructions used SPSS version 26.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.


Extracellular vesicles are lipid bilayer-delimited particles carrying proteins, lipids, and small RNAs. Previous studies have demonstrated that they had regulatory functions both physiologically and pathologically. However, information remains inadequate on extracellular vesicles from the anterior pituitary, a key endocrine organ in animals and humans. In this study, we separated and identified extracellular vesicles from the anterior pituitary of the Duroc swine model. Total RNA was extracted and RNA-seq was performed, followed by a comprehensive analysis of miRNAs, lncRNAs, and circRNAs. Resultantly, we obtained 416 miRNAs, 16,232 lncRNAs, and 495 circRNAs. Furthermore, GO and KEGG enrichment analysis showed that the ncRNAs in extracellular vesicles may participate in regulating intracellular signal transduction, cellular component organization or biogenesis, small molecule binding, and transferase activity. The cross-talk between them also suggested that they may play an important role in the signaling process and biological regulation. This is the first report of ncRNA data in the anterior pituitary extracellular vesicles from the duroc swine breed, which is a fundamental resource for exploring detailed functions of extracellular vesicles from the anterior pituitary. The pituitary gland is often regarded as the “master gland”, coordinating the complex functions of multiple endocrine glands along with the hypothalamus . The anthttp://www.Exocarta.org). EVs were previously considered to be a waste of protein produced during cell metabolism (Extracellular vesicles (EVs) are a type of nano-scale vesicles that can be secreted by most eukaryotic cells . EVs usutabolism  until retabolism . More antabolism . Recentltabolism .Non-coding RNA (ncRNA) is a variety of functional RNA molecules that would not be translated into proteins. MiRNA is a type of small ncRNA and can negatively regulate the expression of its target gene expression at the post-transcriptional level . MiRNAs As an important endocrine organ, there were very limited information about the secretion of EVs in the anterior pituitary gland. In this study, we extracted and identified EVs from the anterior pituitary of Duroc swine breed for the first time, and we also explored its ncRNAs. This study will provide a basis for further exploration of the functions of pituitary EVs.3 pieces and cultured in serum-free Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12)  supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin . Forty-eight hours later, the conditioned media (CM) was harvested and centrifuged at 300×g for 10 min to pellet debris and cells. The supernatant was transferred to a fresh tube, and EVs were isolated using an Exoquick EV Isolation Kit  according to the manufacturer’s instructions as described previously  at 60 days of age, which were purchased from the Jintuan farm of JIADA GROUP . An endotracheal tube  was used to anesthetize the pigs with isoflurane  . Then, teviously , and theA drop of EV suspension (about 10 µL) was fixed on a formvar-coated copper grid for 2 min, washed briefly in ultrapure water, negatively stained with 1% uranyl acetate, and observed by transmission electron microscopy  at an acceleration voltage of 80 kV.The size distribution of EVs was analyzed by tracking particles and sized automatically based on Brownian motion and the diffusion coefficient using Zetasizer  at 25°C.Total protein content was assayed using the Pierce BCA Protein Assay Kit  according to the manufacturer’s instructions. The proteins were measured using a FluorChem M Fluorescent Imaging System , separated by SDS-PAGE (10%), and transferred to a polyvinylidene difluoride membrane . We used three positive markers  for Western blots. After blocking with 5% skim milk for 2 h, the membranes were incubated overnight at 4°C with specific antibodies against CD9, CD63 , and TSG101 . We applied horseradish peroxidase–conjugated goat anti-rabbit IgG  as a secondary antibody for 1 h at room temperature.® Multiplex Small RNA Library Prep Set for Illumina® . After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2,500/2000 platform and 50 bp single-end reads were generated at the Novogene Bioinformatics Institute . A total amount of 2 μg RNA per sample was used as input material for the Library preparation for lncRNA sequencing. The libraries were sequenced on an Illumina Hiseq 2,500 platform, and 125 bp paired-end reads were generated. The circRNA in the whole transcriptome project was analyzed in lncRNA sequencing, and no separate library was built.We extracted total RNA from EV suspension samples using Trizol reagent  according to the manufacturer’s instruction. RNA quantity and quality were assessed using an RNA 6000 Nano Lab-Chip Kit and Agilent 2,100 Bioanalyzer  with RNA integrity number >7.0. A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNextRNA was extracted from EVs using Trizol reagent, and RNA concentration was detected by a spectrophotometer . Total RNA (1 µg) was reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). U6 was used as control. The 2-∆∆CT method was applied to determine relative miRNA expression levels.http://srna-tools.cmp.uea.ac.uk/) were used to obtain the potential miRNA and draw secondary structures. The characteristics of the hairpin structure of miRNA precursor can be used to predict novel miRNA. The available software miREvo , 15668033 (EV_2), and 15353011 (EV_3) clean reads that were screened from small RNA (sRNA) for subsequent analysis after quality evaluation , growth hormone-releasing hormone receptor (GHRHR), prolactin-releasing hormone receptor (PRLHR), follicle-stimulating hormone subunit beta (FSHB), and luteinizing hormone subunit beta (LHB)  . Some ot binding . KEGG pa binding . These dAfter evaluating the data output quality, we obtained 495 novel circRNAs , growth hormone receptor (GHR), prolactin receptor (PRLR), gonadotropin-releasing hormone receptor (GNRHR), and POU class 1 homeobox 1 (POU1F1) (Recent studies suggested that lncRNAs could function as endogenous miRNA sponges to prevent miRNA from binding to reduce the regulatory effect of miRNAs on their target mRNA . To furt(POU1F1) . We also(POU1F1) . Its annGH1, POMC, GHR, GHRHR, PRLR, LHB, PRLHR, FSHB, GNRHR, and POU1F1  expression (ZEB1 (Firstly, considering that the pig pituitary is small and difficult to obtain, we used the Exoquick Isolation Kit to isolate the EVs, then identified them using transmission electron microscopy and western blot detection of CD9, CD63, TSG101, and Calnexin, followed by RNA extraction and sequencing. The Venn diagrams of miRNAs, lncRNAs, and circRNAs were drawn through analysis to show the distributions of numbers among three samples (additional  in pigs . MiR-375pression . Let-7f-pression . Mir-26apression . MiR-200on (ZEB1 . KEGG anCeRNAs regulate gene expression by competitively binding to microRNAs . Recent GH1, GHRHR, PRLHR, FSHB, and LHB. Some other lncRNAs could co-express with genes involved in EVs’ marker protein, protein transport, and docking such as CD63, CD81, TSG101, Rab27A, Rab27B, and UBL3. On the other hand, the signal of a two-circRNA was found to be able to predict tumor recurrence in clinically non-functioning pituitary adenoma (There are a large number of studies that have identified the role of lncRNA in pituitary function. Researches have shown that the anterior pituitary lncRNA of rats plays an important role in hormone and reproduction development and regulation . MIR205H adenoma . Another adenoma . RegardiOn the whole, our study is the first exploration of the expression of ncRNAs in EVs delivered by the anterior pituitary in Duroc swine model. MiRNAs, lncRNAs, and circRNAs of EVs from the anterior pituitary may act as novel regulators of pituitary development and endocrine regulation. These findings provided an insight into EVs derived from the anterior pituitary and are helpful to explore the potential functions of EV cargoes.


Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation. Pre-mRNA is synthesized in the nucleus, where it undergoes capping/polyadenylation and splicing, as well as several post-transcriptional modifications. The mRNA is then transported out of the nucleus to the cytoplasm, where it is translated into protein and subsequently degraded . This whThree main RNA processing MoA emerged that took antisense technologies to the clinical stage: (1) RNase H-dependent mRNA degradation with gapmers, (2) RNA interference for siRNA-mediated degradation of transcripts, and (3) splicing modulation via steric blocking AONs. These MoA generated several FDA-approved drugs in recent years ,9 and haThe emerging field of epitranscriptomics has enabled the identification of more than 170 nucleotide RNA modifications , offerinWe have come a long way to successfully translate the early discoveries in oligonucleotide therapeutics into proof-of-concept in human trials with a meaningful impact on patients’ lives. One of the biggest challenges has been the efficient delivery of oligonucleotides to the site of action . Here, we summarize the leading platform technologies or administration procedures that enabled these macromolecules to reach the site of action and hybridize to their target RNA in a highly sequence-specific manner .Therapeutic oligonucleotides usually have chemical modifications in their phosphate backbone and sugar rings . The mos5C) . . 162]. IThe inosine RNA modification has attracted much attention over the years ,171,172.The potential for inosines to recode transcripts and affect events such as splicing or translation has generated wide interest from the academic community and the biopharma industry  to correMore recently, the Stafforst’s lab could execute this concept with chemically modified guide RNA oligos , suggestendo conformation [Pseudouridine (Ψ) is the most abundant modified nucleotide found in RNAs. Ψ is derived from uridine via pseudouridylation, a base-specific isomerization reaction, where the N1-C1′ is broken, and a new C5-C1′ bond is established A. Thereformation ,194. Ψ cormation ,196,197.ormation ,199 , oligonucleotide design, and improvements in the chemical modifications have provided approved treatments for diseases with a high unmet medical need. Although only the most well-known MoA have so far succeeded in the clinics, a new generation of antisense-epitranscriptomic therapies is in the pipeline. The current momentum in the study of RNA modifications and the field of epitranscriptomics is sparking a strong interest in taking the lessons from antisense therapies to discover and develop new drugs based on novel MoA. The door is now open to further developments aimed to repair single point mutations while harnessing endogenous epitranscriptomics machinery.The technologies developed and breakthroughs achieved for the antisense mechanisms, such as efficient large-scale manufacturing, next-generation chemical modifications, new conjugates that improve cellular uptake, and the revolution provided by LNP formulations, are now becoming enabling technologies for the new epitranscriptomic-based MoA. The development of new preclinical platforms, such as the use of patient-derived materials , as a complement or an alternative (when there is a lack of) to animal models holds great promise for validation of RNA editing modalities.Alongside with CRISPR editing technologies ,213,214,


RNA molecules can fold into complex structures and interact with trans-acting factors to control their biology. Recent methods have been focused on developing novel tools to measure RNA structure transcriptome-wide, but their utility to study and predict RNA-protein interactions or RNA processing has been limited thus far. Here, we extend these studies with the first transcriptome-wide mapping method for cataloging RNA solvent accessibility, icLASER. By combining solvent accessibility (icLASER) with RNA flexibility (icSHAPE) data, we efficiently predict RNA-protein interactions transcriptome-wide and catalog RNA polyadenylation sites by RNA structure alone. These studies showcase the power of designing novel chemical approaches to studying RNA biology. Further, our study exemplifies merging complementary methods to measure RNA structure inside cells and its utility for predicting transcriptome-wide interactions that are critical for control of and regulation by RNA structure. We envision such approaches can be applied to studying different cell types or cells under varying conditions, using RNA structure and footprinting to characterize cellular interactions and processing involving RNA. Precise structural conformations are a hallmark of functional nucleic acid polymers in cells. For example, the genome is arranged in a compact three-dimensional structure, whose dynamics and solvent accessibility are critical for the control of gene expression . MethodsThe ability of RNA molecules to fold into complex two- and three-dimensional structures is critical for the many biological functions they perform ,5. CurreTransferring conventional chemical probes from one-RNA-at-a-time measurements to transcriptome-wide scale has proven to be a formidable challenge. Bifunctional RNA structure probing reagents that enable reactivity with RNA and enrichment of reaction sites has been demonstrated to dramatically increase signal-to-noise ratios of structural information per read arising from chemical adducts ,11. We pLight Activated Structural Examination of RNA 18s Primer 1 T. tencongensis was designed into a plasmid with IDT. The plasmid encoding the SAM-I sequence was transformed into a One Shot Top 10 chemically competent cells (ThermoFisher) and plated on lysogeny broth  supplemented with 100 mg/ml ampicillin (VWR) agar plates. A single colony was selected in a 3 ml culture and grown overnight. The resulting plasmid was isolated using a QIAprep Miniprep kit (Qiagen). Transcription template was prepared by PCR in 50 μl volumes using primers directed against the T7 promoter  and an adaptor sequence for reverse transcription  with Phusion High-Fidelity PCR master mix (NEB). The mixture was treated with 1 μl of DPN1 (NEB) restriction enzyme and incubated at 37°C for 30 min. The resulting PCR product was purified using a QIAquick PCR purification kit (Qiagen). In vitro transcription of SAM-I RNA was performed in 20 μl reactions using 800 ng of template SAM-I DNA with a T7 Ribomax transcription kit (Promega). Samples were treated with 1 μl of RQ1 DNAse (Promega) at 37°C for 30 min. The resulting solution was brought up to 100 μl and precipitated with 10 μl of 3 M NaOAc (pH 5.2), 1 μl glycoblue (ThermoFisher), and 300 μl ethanol. The precipitated RNA stored at −80°C for 30 min, centrifuged at 15 000 rpm for 15 minutes, and resuspended in 50 μl of RNase-free water. An aliquot of SAM-I was run on a denaturing PAGE gel  alongside a 100 bp RNA ladder (Invitrogen). The band of interest was visualized with 1× SYBR-gold stain (ThermoFisher) in water for 15 min. Resulting concentrations of RNA was quantified by integrating intensity of the ladder with the RNA band of interest.A 94 nucleotide construct consisting of the sequence for the SAM-I riboswitch from the metF-metH2 operon of 32P-ATP, 1 μl T4 PNK, NEB). The reaction was allowed to proceed for 2 h at 37°C. Reactions were stopped by the addition of 5 μl of Gel Loading Buffer II. The reaction was loaded onto a 15% denaturing PAGE gel. The band of interest was visualized by a phosphorimager . The resulting band was excised and eluted overnight in 400 μl of 300 mM KCl. Resulting solution was EtOH precipitated and dissolved to 8000 counts per minute (cpm)/μl for further use in reverse transcription.Primer DNA was 5′ end labeled in 10 μl reactions in a T4 PNK mix  was added, and the RNA was allowed to equilibrate at 37°C for 5 min. To this mixture 1 μl of 3 M NAz, 1 M NAz-N3 in DMSO (+) or DMSO (−) was added. Reactions were then exposed to 20 W lamp (Zilla Desert UVB 50) UV light for 3 min for NAz, and 10 minutes for NAz-N3. Reactions were brought up to 200 μl water, and extracted once with 200 μl acid phenol/chloroform/isoamyl alcohol , and washed twice with 200 μl chloroform (Sigma). Samples were precipitated by adding 20 μl 3 M NaOAc (pH 5.2), 1 μl glycoblue (20 μg/μl) and 600 μl EtOH. Pellets were washed twice with 70% cold ethanol and resuspended in 5 μl nuclease free water.5 μg total RNA (isolated from HeLa cells) or 10 pmol of In a reaction volume of 50 μl, modified RNA (10 pmol) was incubated with 5 μl dibenzocyclooctyne–PEG4–biotin  for 1 h at 37°C in 1× PBS. Reactions were brought up to 200 μl and extracted once with 200 μl acid phenol/chloroform/isoamyl alcohol (pH 4.5), and washed twice with 200 μl chloroform. Samples were precipitated by adding 20 μl of 3 M NaOAc (pH 5.2), 1 μl glycoblue (20 μg/μl) and 600 μl EtOH. Pellets were washed twice with 70% cold ethanol and resuspended in 2 μl nuclease free water for dot blot analysis, or 10 μl nuclease free water for streptavidin enrichment assays.2HPO4, 8 mM NaH2PO4, 170 mM SDS) for 30 min. To the membrane was added 1 μl Pierce high sensitivity streptavidin-HRP(ThermoFisher) in blocking solution. The membrane was washed twice with wash buffer A (1:10 blocking solution) for 30 min, and twice with wash buffer B  for 5 min. Membrane was incubated with Pierce western blotting substrate and visualized on the ChemiDoc (Biorad) under chemiluminesence hi sensitivity. To visualize RNA, the membrane was stained with a methylene blue solution .Hybond N + membranes (GE) were pre-incubated in 10× SSC. Precipitated biotinylated total RNA was dissolved in 2 μl of RNase free water. RNA was loaded onto the Hybond membrane and crosslinked using 254 nm ultraviolet light. The membrane was incubated with blocking solution  culture medium supplemented with 10% FBS (SAFC) and 1% penicillin streptomycin (Life technologies). K562 cells were grown in RPMI 1640 supplemented with 10% FBS, and 1% penicillin streptomycin. Cells were washed three times with Dulbecco's phosphate-buffered saline  and centrifuged at 1000 RPM for 5 min. Cells (∼3–6 × 107) were resuspended in 45 μl DPBS. 5 μl DMSO (−), 10% final concentration, 5 μl 3 M NAz or 1 M NAz-N3 in DMSO (+) was added to the desired final concentration. Cell suspensions were subjected to UV light for 5 min using NAz and 10 minutes with NAz-N3. Cells were pelleted by centrifugation at 1000 RPM for 5 min and resuspended in 1 ml Trizol Reagent (ThermoFisher). RNA was harvested using Trizol Reagent following the manufacturer's instructions. 500 μl of aqueous phase was then precipitated with 500 μl isopropyl alcohol at room temperature for 10 min. Samples were centrifugated at 15 000 RPM at 4°C and washed twice with cold 70% EtOH.HeLa cells were grown in DMEM . Samples were eluted in 6 μl of RNase-free water and used for subsequent reverse transcription.In 700 μl reaction volume, 50 pmol of biotinylated RNA was added with 50 μl of prewashed Dynabeads MyOne C1 beads (ThermoFisher). The solution was then mixed at room temperature for 1 h. The beads were collected on a magnetic plate and flowthrough was saved. The beads were then washed three times with 700 μl of Biotin Wash buffer . The first wash was saved and combined with the flowthrough for further analysis. Samples were later washed twice with RNase-free water. NAz-N32P-end-labeled DNA primers were annealed to modified RNA by incubating at 95°C for two minutes, then 25°C for 2 min, and 4°C for 2 min. To the reaction, 1 μl of 5× First strand buffer, 0.5 μl nuclease free water, 0.5 μl 100 mM DTT, and 0.5 μl 10 mM dNTP’s were added. The reaction was preincubated at 52°C for 1 min, then 1 μl superscript III (ThermoFisher) was added. Extensions were performed for 15 min. To the reaction, 1 μl 4 M sodium hydroxide was added and allowed to react for 5 min at 95°C. The resulting complementary DNA (cDNA) was snap cooled on ice, and ethanol precipitated according to above procedures. Purified cDNA was resuspended in 2 μl of nuclease-free water and 2 μl of Gel Loading Buffer II. cDNA products were resolved on 10% denaturing polyacrylamide gel, and visualized by a gel imager .3, NAz-N3 or DMSO, RNA extracted, and coupled to DBCO-biotin, as described previously  and brought back up 9 μl water. RNA was fragmented by adding 1 μl of Fragmentation Reagent (Ambion) at 70°C for 30 s and then stopped by adding 1 μl of RNA Fragmentation Stop Solution (Ambion) and moved to ice. RNA was desalted with a Zymo RNA clean and concentrator as above and again dried with a lyophilizer. End repair occurred by adding 5 μl of water, 2 μl of 5x PNK buffer , 0.5 μl SUPERaseIn (Thermo Fisher Scientific), and 1.5 μl of T4 PNK (NEB) and 1.5 μl Fast AP (Thermo Fisher Scientific). These reactions were incubated at 37°C for 45 min. After end-repair, 1 μl of 2 μM 3′ pre-adenylated adaptor (3′biotin linker for DMSO or 3′dideoxycytosine linker for NAI-N3 or NAz-N3), 1 μl of 10× T4 RNA Ligase I buffer (NEB), 1 μl T4 RNA Ligase, High Concentration (NEB), 1 μl of 100 mM DTT and 6 μl of 50% PEG8000 were added. Ligation reactions were incubated at 25°C for 6 h. Excess, unligated adaptors were destroyed enzymatically by adding 3 μl of 10× NEB Buffer 2 (NEB), 2 μl Rec Jf (NEB), 1 μl 5′ deadenylase (NEB), 4 μl of water and incubate at 37°C for 1 h. Ligated RNA products were desalted and removed of enzymes with the Zymo RNA clean and concentrator.RNA samples, typically 100 ng for DMSO and 1000 ng for NAI-N3, NAz-N3 modifications to the RNA) were captured by addition of 5 μl of MyOne Streptavidin C1 Dynabeads (ThermoFisher Scientific) that had been rinsed and suspended in 50 μl of Biotin-IP buffer , and rotation for 45 min at 25°C. Non-biotinylated RNA was washed away with five additional washes of Biotin-IP buffer at 25°C and then excess detergent and salt were reduced by two bead rinses in NT2 buffer . Beads were resuspended in 12 μl water 1 μl of 3 μM RT primer (Biotinylated RNAs (via 3′-biotin linker for DMSO libraries or NAI-NT primer  and 1 μlEluted cDNA was transferred to a new PCR tube containing 15 μl of 2X Phusion HF-PCR Master Mix (NEB), 0.5 μl of 30 μM P3/P6 PCR1 oligo mix  and 0.5 10 μg total RNA extracted with Trizol (Ambion) was fragmented with fragmentation reagent (Ambion) at 70°C for 10 min followed by precipitation with ethanol. Reverse transcription was performed with PASSEQ7-2 RT oligo:[phos]NNNNAGATCGGAAGAGCGTCGTGTTCGGATCCATTAGGATCCGAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTVNand Superscript III (Invitrogen). cDNA was recovered by ethanol precipitation and 120–200 nucleotides of cDNA was gel-purified from 8% urea–PAGE. Recovered cDNA was circularized with Circligase™ II (Epicentre) at 60°C overnight. Buffer E (Promega) was added to the cDNA and heated at 95°C for 2 min, and then cool to 37°C slowly. Circularized cDNA was linearized by BamH I (Promega). cDNA was collected by centrifugation after ethanol precipitation. PCR was carried out with primers PE1.0 and PE2.0 containing index (Illumina). Around 200 bp of PCR products was gel-purified and submitted for sequencing (single read 100 nucleotides).Raw sequence reads were quality-checked using FastQC  and demuhttps://doi.org/10.1101/179648) for each protein were located in each peak; peaks lacking the motif were discarded. icLASER enrichment scores were extracted for each motif position, plus five basepairs up- and downstream from the motif. Negative control sites were identified as occurrences of the same 5 bp motif that fall outside of eCLIP peaks. As with binding sites, icLASER enrichment scores were extracted for a 15 bp range centered on the motif. A number of negative sites equal to the number of positive sites was selected randomly from the pool of possible negatives.Publicly available eCLIP peak data for five RNA binding proteins in K562 cells was downloaded from the ENCODE project . Known 5icLASER analysis at poly-A signal sequences was conducted similarly; however, Ensembl-annotated polyadenylation signal sequences were used to identify positive sites, and enrichment scores were retrieved for a range of 20 bp up- and downstream of the 6 bp polyadenylation signal motif.in vitro and in vivo data sets were divided in half at random; half for the data was used to train the SVM, while the remaining half was used to test its predictions. icLASER scores at each position in the 15bp range surrounding the motif of interest were input as features. Ranges containing null values for icSHAPE or icLASER enrichment scores were discarded.SVMs were implemented using libSVM . Both trSVM analysis was conducted as with RNA-binding proteins. icSHAPE or icLASER scores for a 46 bp range centered on the polyadenylation signal sequence were used as features.in vivo click LASER, icLASER). Towards the goal of integrating multiple, orthogonal measurements of RNA structure, we set out to directly compare icSHAPE  and icLASER (solvent accessibility) and use these measurements to predict a verity of functional element with RNAs across the transcriptome   Figure  and D3, which we predicted would be able to undergo light transformation to the activated nitrenium ion, would also preserve the alkyl azide for Cu-free ‘click’ after RNA adduct formation. Notably, aroyl azides are amenable to activation by lower energy long wavelength light, whereas alkyl azides need higher energy short wavelength light for activation  would optimally not reduce selectivity or efficiency of NAz in measuring nucleobase solvent accessibility. As such, NAz-Ntivation . We hypog Figure .3 and compounds 1 and 2  long wavelength UV light. Following irradiation, we isolated the RNA and incubated the RNA with DBCO-biotin. We observed biotin conjugation on a streptavidin dot blot only in the sample incubated with long wavelength UV light and NAz-N3 . We used NAz-N3 modified riboswitch RNA, biotinylated the RNA using SPAAC, and subjected it to streptavidin-coated magnetic bead enrichment. Biotinylated RNA was then eluted and compared against input RNA. Each sample was then reverse transcribed with a 32P-cDNA primer and analyzed by denaturing gel electrophoresis. As shown in Figure 3 enriched samples displayed significantly higher signal for structure stop cDNA and dramatically reduced full-length extensions. Finally, NAz-N3 elution mapped onto sites of modification similar observed with NAz  and K562 total RNA isolated and re-folded outside of cells (in vitro) in independent experiments with NAz-N3 (icLASER) and NAI-N3 (icSHAPE). We applied our well-established protocol for the isolation of poly-adenylated (polyA) RNA and mapping of biotin-conjugated RNA structure probes through deep sequencing .,26.3 protivation . Transcrtivation  and sugg3 and a solvent exposed C8 of purines reacts with the nitrenium ion of NAz-N3. We interpret both icSHAPE and icLASER RT stops as a residue that is flexible and solvent exposed, which often occurs in loop regions of RNA structural elements. A site that is not reactive to icSHAPE, but is reactive to icLASER, is a site that is not flexible, but is solvent exposed. Correspondingly, a site that is reactive to icSHAPE, but not icLASER, is a site that is flexible, but not solvent exposed. We have observed many sites that are reactive to both SHAPE and LASER electrophiles as well as those that have orthogonal reactivity; these reactivities are controlled by the local environment of the nucleobase  present in the untranslated region of some messenger RNAs and which binds proteins, modulating the flexibility and solvent accessibility of its nucleotides ,32. In Ks Figure  and D . Specif are high resolution at the site of interaction but must be done one RBP at a time and do not directly report on the structural features of their binding sites ,39. ConvGiven the orthogonality of icLASER and icSHAPE, we hypothesized that integrating their reactivity profiles simply from an in vivo experiment could provide a robust strategy to predict RNA-protein interactions Figure . We usedTo develop initial conceptual rules for this strategy, we started with focused analysis on four RBPs with published structural data (X-ray or NMR) and selected TRA2A, RBFOX2, QKI and PUM2. TRA2A Figure  and C isRBFOX2 is an RBP that plays important roles in regulating RNA stability through interactions in the 3′-UTRs of mRNAs . RBFOX2 Quaking1 (QK1) is an RNA binding protein that is important for regulating mRNA trafficking and stability . Figure and G ic2), which is related to SHAPE reactivity . Co. Co46). activity . icLASERThe four examples detailed above suggest to that differences in icSHAPE and icLASER signal, at the same primary sequence motif, differ in a manner related to their being bound by RBPs in living cells (eCLIP bound vs not bound). To generalize this observation, we implemented a Support Vector Machine (SVM) algorithm to learn icSHAPE and icLASER signals in an effort to predict sites of RBP binding. We evaluated performance of the SVM using the area under the receiver operating characteristic (ROC) curve for each protein Figure . Using tOverall, these results support the notions that: (i) RNA solvent accessibility (icLASER) and RNA flexibility (icSHAPE) probing can be utilized to predict RNA-protein interactions, (ii) icLASER and icSHAPE data can be utilized to complement CLIP datasets to further support protein occupancies determined by orthogonal methods and (iii) by combining icLASER and icSHAPE, robust and transcriptome-wide predictions of many RBPs is possible without protein-centric techniques such as CLIP. We suspect the robust, but in some instances imperfect prediction of RBP binding sites may be due to the inability of icLASER and icSHAPE probing to differentiate changes in (A) protein binding that alter icSHAPE and icLASER reactivity from (B) changes in RNA structure that cannot be accounted for in all instances. Despite this, our SVM was trained only on 8 physical experiments  while the data for the 75 eCLIP profiles resulted from over 300 individual experiments. Thus, chemical probing of RNA reactivities offers a simplified and multi-RBP approach to define binding sites in living cells.Having demonstrated the utility of combining icLASER and icSHAPE reactivities in predicting actively bound RBP sites across the transcriptome, we text if this strategy could similarly predict another class of RNA functional elements. One important set of elements are the polyadenylation signals (PAS) that is controlled through protein recognition of a specific motif, resulting in cleavage and polyadenylation ,49. NearTo experimentally determine PAS sites, for SVM analysis, we generated the first polyadenylation sequencing data (PAS-seq ) for K563, similarly relies on specific photoactivation of an aroyl azide by long-wavelength UV light, but can subsequently be ligated to biotin using copper-free ‘click’ chemistry, for enrichment of modified sites of adduct formation. Using this chemistry, we measured RNA solvent accessibility in K562 cells, transcriptome-wide.Here, by developing novel RNA structure bi-functional probes, we extend the utility and flexibility of our previously reported RNA solvent accessibility probe, NAz. The new reagent, NAz-NWith the development of transcriptome-wide RNA structure probing techniques, an exciting but thus far poorly explored aspect of these data has been the possibility to infer interactions between RNAs and their cellular partners. By employing a computational strategy (SVM) to combine icSHAPE and icLASER (as well as other reactivity-based measurements) we take a critical step towards learning the potential of these methods in predicting RBP binding and other functional RNA processing activities like PAS selection. We demonstrate the power of this approach by predicting RNA-protein binding sites for a large number of RNA binding proteins. We demonstrate that such an approach could be very powerful for measuring RNA-protein interfaces and is highly complementary with protein-centric methods such as eCLIP. Further, we utilize RNA structure probing to identify a structure signature associated with polyadenylation sites, which is due to the presence of a protein bound at the PAS site. This extension also enables structure probing to be utilized for other aspects of RNA biology, such as RNA processing.The complexity of nucleic acid polymer structure is now well appreciated for DNA and the folding of the genome; importantly, measuring the structure of such nucleic acids and how proteins interact with DNA has been incredibly valuable for understanding how the genome is regulated to control biological processes within cells. Our combined icSHAPE-icLASER strategy make headway toward this goal for RNA, by the continued development of tools for transcriptome-wide measurement of RNA structure and RNA interactions that can contribute to its biological function and regulation. We anticipate that icLASER (a new aspect of transcriptome-wide RNA structure probing via solvent accessibility) will become an increasingly useful chemical tool to probe RNA structure in living cells transcriptome-wide. By demonstrating the predictive power of these tools, we expect RNA structure probing to expand its value into aspects of RNA biology.icLASER and icSHAPE datasets are deposited on GEO under the accession number GSE132099.Additionally, icSHAPE datasets are deposited on the ENCODE database under experiments ENCSR976RFC, ENCSR803XFA, ENCSR286LXS, ENCSR992XHC, ENCSR052BBY, and ENCSR836VQU.PAS sequencing datasets are deposited on GEO under the accession number GSE145400.gkab885_Supplemental_FilesClick here for additional data file.


Drawing on cognitive-affective system theory, this study proposes that employees’ perceived green human resource management (HRM) influences their’ workplace green behaviors through two psychological processes: the cognitive and the affective route. By analysing 358 questionnaires collected from Chinese firms in the oil and mining industry, we obtain evidence in support of our predictions, finding that employees’ perceived green HRM positively impacts their voluntary workplace green behaviors and green creativity. Additionally, green psychological climate and harmonious environmental passion are found to partially mediate the relationship between employees’ perceived green HRM and voluntary workplace green behavior while harmonious environmental passion is found to fully mediate the relationship between employees’ perceived green HRM and green creativity. These findings shed light on the importance of green HRM in shaping employees’ proactive workplace green behaviors and uncover how green HRM transforms employees’ cognitive, affective, and motivational (CAM) factors into green actions. At present, economic development has inevitably brought about issues related to environmental pollution in China, including climate change, sewage discharge, and biodiversity loss. Such environmental issues are directly or indirectly caused by people’s daily behaviors . As orgaThis paper focuses on employees’ proactive workplace green behaviors . Voluntary workplace green behavior refers to “discretionary employee actions that contribute to the environmental sustainability of the employer organization but are not under the control of any formal environmental management policies or system”  (p. 3). This study intends to make three main theoretical contributions. First, it adds to the literature of green HRM by exploring the employee outcomes of green HRM in the workplace, thus help better understand the concept of green HRM as well as its consequences. As an emerging research topic of HRM, green HRM has not been sufficiently explored in terms of its effect on employees’ workplace outcomes. Even though past research mostly reached the conceptualization stage ,25, empiSecond, past research suggests that HRM practices might not directly exert influence on employee outcomes in the workplace, but indirectly does so through certain path or some psychological processes . HoweverLast but not least, existing research explained the influence mechanism of green HRM on employees from various theoretical perspectives, such as the theory of planned behavior (TPB) , supplieThe remainder of this paper is organized as follows. First, we develop our hypothesis based on cognitive-affective system theory. We then conduct an empirical study from the oil and mining industry and discuss the results, while detailing the associated methods. From there, we present the theoretical contributions and practical implications of this research. Finally, we summarize the current study by outlining its limitations and potential future research directions.Cognitive-affective system theory was first used to describe the dynamics between individuals’ personalities and related behaviors . It statAccording to this theory, the personality system contains mental representations whose activation leads to the behavioral consistencies that characterize the person. These representations consist of diverse cognitive-affective units, which include the person’s construal and representations of the self, people, and situations, enduring goals, expectations, beliefs, and feeling states, as well as memories of people and past events . DrawingThis paper measures green HRM from employees’ perceptions because differences in personal experiences and idiosyncratic preferences may lead employees to react differently to practices ,37. ExisHypothesis 1a.Employees’ perceived green HRM is positively related to employees’ voluntary workplace green behavior.Hypothesis 1b.Employees’ perceived green HRM is positively related to employees’ green creativity.In this study, we draw from cognitive-affective system theory and propose that employees’ perceptions of green HRM may transmit its influence on employees’ proactive green behaviors through two different paths . Cognitive attributes such as expectancies influence how individuals perceive cues and construct meaning of green HRM . In partEmployees’ social interaction with their organizations will contribute to the psychological climate, and they can determine the value of organizational practices, procedures and policies that they observe or encounter , includiWe argue that the green psychological climate perceived by employees could increase their willingness to make extra efforts beyond their duties. Prior research  suggestsHypothesis 2a.Green psychological climate mediates the relationship between employees’ perceived green HRM and employees’ voluntary workplace green behavior.Hypothesis 2b.Green psychological climate mediates the relationship between employees’ perceived green HRM and employees’ green creativity.In addition to the cognitive mediating process of green psychological climates, employees’ perceived green HRM may influence employees’ green behaviors via an affective route. Affect refers to a broad range of feelings that individuals experience including emotions (intense feelings that are directed at green HRM), moods , and attitudes (positive or negative orientations toward green HRM) ,56. BaseHarmonious passion consists of two key characteristics: the activity that one feels harmoniously passionate about is autonomously integrated into one’s identity and is enjoyable to oneself . EmployeHypothesis 3a.Harmonious environmental passion mediates the relationship between employees’ perceived green HRM and employees’ voluntary workplace green behavior.Hypothesis 3b.Harmonious environmental passion mediates the relationship between employees’ perceived green HRM and employees’ green creativity.In sum, the hypothesis model is shown in The sample of firms examined in this study was drawn from the oil and mining industries in China. Compared to other industries, companies of the oil and mining industries generally pose a greater threat to the environment . The oilA cover letter attached with the questionnaire assured the participants that their participation was voluntary and their responses would be used only for research purposes . To reduFor the initial survey, we distributed 500 questionnaires, and 100 individuals failed to respond to our survey invitations. After removing incomplete questionnaires, we obtained 358 valid questionnaires yielding a response rate of 71.60%. The majority (57.82%) of the respondents were males. Of the respondents, 19.83% were under 25 years old, 22.90% were 26–35 years old, 22.35% were 36–45 years old, and 34.92% were more than 46 years old. Regarding their work experience, 10.90% had less than 5 years, 21.23% had 5–10 years, 32.12% had 11–15 years, and 35.75% had more than 16 years. Regarding their education level, 16.20% had a secondary technical school degree, 26.26% had a junior college degree, 31.56% had a Bachelor’s degree, and 25.98% had a Master’s degree. As to their occupations, 33.16% engaged in mining work, 21.08% engaged in geological prospecting work, 27.91% engaged in oil and gas field development, and 17.85% engaged in petroleum refining work.We followed the standard translation and back-translation procedures  to ensurPerceived green HRM was assessed with items taken from Tang et al.  see App. The 18-Green psychological climates were assessed with five items taken from Norton et al.  see App. Each paHarmonious environmental passion was assessed with the 10-item scale developed by Robertson and Barling  see App. Sample Voluntary workplace green behavior was assessed with a measurement developed by Kim et al.  see App. SuperviGreen creativity was measured with the 6-item scale developed by Chen and Chang  see App. SuperviControl variables included each employee’s gender, age, years of work experience and education level because this demographic information may influence individual’s attitude and behavior towards environmental issues based on previous research ,29.2/df = 1.93, RMSEA (Root Mean Square Error of Approximation) = 0.05, CFI (Comparative Fit Index) = 0.90, IFI  = 0.90). p < 0.01; r = 48, p < 0.01; r = 33, p < 0.01; r = 24, p < 0.01, respectively).We applied the PROCESS method  to test We also applied the PROCESS method  to test Besides, combining The main purpose of this study was to examine the relationship between employees’ perceived green HRM and their proactive outcomes (including voluntary workplace green behavior and green creativity) and to explore underlying mechanisms. In line with our expectations, employees’ perceived green HRM positively impacts their voluntary workplace green behavior and green creativity. This finding is in line with past research showing that green HRM has a direct impact on employee outcomes ,24,26. MThe findings of this study make important contributions to the literature in three respects. First, past research suggests that HRM practices might not directly influence employee outcomes in the workplace but may indirectly do so through certain paths or psychological processes . DespiteAs a key practical implication of this study, we find that employees’ perceived green HRM may help organizations stimulate their proactive environmentally oriented behavior. To achieve an organization’s green goals and elicit broader positive employees’ attitudes and behaviors, organizations should hire employees with strong environmental sensibilities and develop training programs to enhance employee skills for effectively undertaking green activities and enhancing green cognition. Organizations should also provide green performance indicators to performance management systems and appraisals. Further, organizations should offer green rewards to employees and involve employees in problem-solving and decision-making regarding green issues. In addition, our findings show that employees’ perceived green HRM influences their outcomes through difference paths. Voluntary workplace green behavior is stimulated by both green psychological climates and harmonious environmental passion. Green creativity is only stimulated by harmonious environmental passion. As green creativity is connected to the maintenance of core competencies of an organization and is very important for firm growth, managerial attention should focus on ways to foster and develop employees’ positive emotions, including their levels of harmonious environmental passion. For example, organizations may cultivate employees’ harmonious environmental passion by strengthening their sense of psychological ownership  and may provide guidance on issues concerning creativity to enhance their employees’ positive views of their organizations.Several limitations of our study can guide future research. Firstly, the data collection process was all carried out in a single location and the data from the oil and mining industry has limited representativeness. As such, it may be necessary to explore how green HRM is implemented in different parts and different industries in China. The present work might be extended to other geographic and industrial settings, both in China and cross-culturally. Secondly, we only tested two mediating effects of cognitive- and affective-based variables on relations between employees’ perceived green HRM and their proactive green behaviors. In order to respond to the call for the development of the psychology of human behaviors and cognitive processes in complex environments , future


Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins , as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay. Penicillium genus cause severe postharvest fruit diseases even when postharvest technologies are applied. Penicillium digitatum, the cause of citrus green mold, and Penicillium italicum, the cause of citrus blue mold, are the main responsible agents of postharvest citrus losses worldwide, since more than 90% of citrus rots are produced by these two species .,14.P. diP. digitatum infection to citrus fruit. PeAfpA, which had been previously described as effective in the control of the fungus in orange fruits [P. digitatum infection. PeAfpA delayed fungal infection throughout the experiment; PAFB did not affect fungal growth at 4 dpi, although it significantly decreased the incidence of infection at 5, 6 and 7 dpi, while NFAP2 failed to control P. digitatum at 7 dpi. The average efficacy at 7 dpi was around 30% and 20% of disease reduction for PeAfpA and PAFB, respectively. In order to confirm the effect of AFPs, a second experiment with AFPs in individual and combined treatments was carried out  than to PAFB. These results show that AFPs act in a species-specific manner.PAFB displayed the highest antifungal potency in in vitro assays with MIC values in the range of 0.12–0.25 μM, comparable to those of PeAfpA 0.15–0.3 μM)  and PdAf–0.3 μM  P. digitatum and P. italicum, whereas the MIC values against P. expansum were much higher. The in vitro growth inhibitory activity of PAFC against opportunistic human pathogenic members of the Candida genus has been recently described [P. expansum class C protein, PeAfpC, which did not show any antifungal activity against the three Penicillium species tested [Byssochlamys has been reported [Penicillium genus.PAFC and NFAP2 showed a moderate antifungal activity against escribed . Remarkas tested . Only rereported . Regardireported . Here, oAspergillus giganteus was the first experimentally tested ascomycetous antifungal protein to control plant and postharvest diseases in vivo. A. giganteus AFP has been reported to successfully control postharvest decay caused by Magnaporthe oryzae in rice [Fusarium oxysporum in tomato plant seeds [Alternaria alternata in banana [Penicillium decay using three pathosystems, P. digitatum-orange fruit, P. italicum-orange fruit and P. expansum-apple fruit. PAFB provoked disease reductions in orange and apple fruits, although with moderate efficacy. In agreement to that observed in the in vitro assays, the PAFB effect in protection experiments was equivalent to that observed with PeAfpA in the side-by-side experiments conducted. We showed previously that PeAfpA exerts protection against P. digitatum in oranges and against P. expansum in apples at concentrations of 0.15–15 µM, although variations in the percentage of disease reduction among experiments, as those observed here, were reported [P. italicum infection to Navel and Lanelate oranges while PeAfpA did not show any effect. This is the first time that PeAfpA is evaluated in the pathosystem P. italicum-orange fruit, and despite its potency against the fungus in in vitro experiments [P. digitatum and P. italicum  might contribute to disease incidence, while fungicides are commercially applied onto the entire fruit surface. In addition, the inoculum dose used in protection assays is aggressive since it renders around 80–100% of infection at 6–7 dpi. Interestingly, when fungicides such as imazalil and thiabendazole are applied in parallel assays with antifungal peptides under similar conditions as those described here, fungicides performed similarly and the average efficacy of disease reduction was not significantly different to that provoked by AFPs or antifungal peptides ,45. FurtP. digitatum infection in orange fruits (P. expansum infection in apples (To reach the level of efficacy provided by conventional fungicides, combinations of alternative approaches of the same or different nature have been proposed . Here, we fruits  and P. en apples , respectn apples c, the laP. chrysogenum AFPs and N. fischeri NFAP2, which show a species-specific inhibition spectrum against the main Penicillium species causing postharvest decay. PAFB, the most active antifungal protein in in vitro susceptibility tests, delays P. digitatum, P. italicum and P. expansum infection in orange and apple fruits. Future efforts are currently directed to optimize the efficacy of PAFB through combinations with different postharvest control strategies.This study provides additional knowledge about the three