Even though the functional role of mRNA molecules is primarily decided by the nucleotide sequence, several properties are determined by secondary structure conformations. Examples of secondary structures include long range interactions, hairpins, R-loops and G-quadruplexes and they are formed through interactions of non-adjacent nucleotides. Here, we discuss advances in our understanding of how secondary structures can impact RNA synthesis, splicing, translation and mRNA half-life. During RNA synthesis, secondary structures determine RNA polymerase II (RNAPII) speed, thereby influencing splicing. Splicing is also determined by RNA binding proteins and their binding rates are modulated by secondary structures. For the initiation of translation, secondary structures can control the choice of translation start site. Here, we highlight the mechanisms by which secondary structures modulate these processes, discuss advances in technologies to detect and study them systematically, and consider the roles of RNA secondary structures in disease. We discuss the consequences of secondary structure formation for splicing and translation, with particular focus in G-quadruplexes, hairpins and long range interactions. We also discuss the contribution of secondary structures in the regulation of mRNA splicing and in translation initiation and discuss the mechanisms involved.mRNAs are essential molecules in the cell as they are key to extracting information stored in the DNA. Although the function of mRNA molecules is primarily determined by the nucleotide sequence, some properties are determined by secondary structures. Secondary structures are defined as distinct features, including hairpins, long range interactions, G-quadruplexes, R-loops and pseudoknots and they are formed as a consequence of the interactions of non-adjacent nucleotides. Their presence can impact various processes involving the mRNA, including synthesis, splicing and translation. Secondary structures are dynamic and can be modulated by multiple proteins, in particular RNA binding proteins (RBPs), and as they cannot be predicted solely from the primary sequence they are challenging to study. Nevertheless, several assays are available for both 2In RNA, intra and intermolecular long-range interactions, including hairpins, pseudoknots, and G-quadruplexes, are commonly observed. Hairpins are composed of a hybridized stem and a single stranded loop a and b ain vitro, while more recent methods can deduce structures in vivoin vitro, and RNA G-quadruplexes have also been visualized in cellulo using a specific antibody A number of methods that probe RNA structures have been developed. Methods such as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)-seq 3S. cerevisiae and S. pombe, folding energy and GC content in the transcription bubble have been correlated with RNA polymerase distribution, and RNA structures within nascent transcripts promote forward translocation of the polymerase and limit back-tracking A variety of features are associated with RNAPII speed. For instance, the presence of introns and the length of the first intron are both positively correlated with RNAPII speed 4Pre-mRNA splicing is a key biological process that enables the removal of introns and the joining of intervening exons, eventually resulting in a mature mRNA molecule. Alternative splicing affects approximately 90–95% of mRNA transcripts in humans Splicing is mediated through the spliceosome complex which recognizes splice signals, the key members being the 5′ splice site (5′ss), the 3′ splice site (3′ss), and the branch point. The recognition of these consensus sequences is commanded by U1 and U2 small nuclear ribonucleoproteins (snRNPs) and other auxiliary protein factors that are involved in early spliceosomal assembly. Since higher-eukaryotic genes are often interrupted by long introns, early spliceosomal complex assembly over exons recognizes both splice sites during a process commonly known as exon definition cis-regulatory elements can be modulated by the presence of RNA structures in nascent transcripts and in mature mRNAs ATE1 gene, where splicing of two mutually exclusive exons is regulated by competing long-range hairpin structures that span up to 30 kB Even though the RNA structural code has been less explored 5During the mRNA lifecycle, RBPs regulate to a significant extent diverse transcriptional and post-transcriptional stages including splicing, transportation, translation, stability and degradation. They bind to pre-mRNA molecules in the nucleus and regulate its maturation and transportation to the cytoplasm where they regulate translation and degradation. The number of proteins that can bind to RNA in humans is estimated to be more than 1,500, adding complexity to all the aforementioned programs in vivo as demonstrated by high-throughput experiments that identify the sites where RBPs bind to endogenous RNAs such as cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq). One possible explanation is that RNA structures provide additional contextual features beyond the primary motif sequences  or CRISPR-based technologies have enabled systematic interrogation of the roles of diverse proteins, such as RBPs, helicases, and topoisomerases 7G-quadruplex sequences are enriched at promoters and they have been extensively studied in this context CD44 gene in vitro RBP binding to mRNA molecules One of the first exemplary cases of RNA G-quadruplex mediated regulation of alternative splicing was found in the hTERT gene, which encodes for the catalytic subunit of the telomerase enzyme, and one of its exon skipping events is promoted by the stabilization of intronic G-quadruplexes The genome-wide effect of RNA G-quadruplex formation over splicing factor binding remains unclear. High-throughput screening of chemical compounds via dual-color splicing reporters has identified two small molecules, emetine and cephaeline, that disrupt RNA G-quadruplex formation 8D. melanogaster for the DSCAM gene, where RNA-RNA interactions, mediated through multiple structures, regulate the selection of exons within arrays of mutually exclusive exons Long range interactions are important for splicing modulation Hairpins may also directly affect exon skipping events by a mechanism known as “looping-out”, whereby inter-intronic base-pairing RNA interactions can loop out exons to promote their skipping FGFR2, the formation of a hairpin structure is required for efficient splicing from two mutually exclusive exons and its splicing effect is not dependent on its primary nucleotide composition as shown using minigene assays The formation of RNA structures can also enhance RBP regulatory range by bringing distal regulatory elements in close proximity with their exon targets The fibronectin EDA exon is controlled by seven hairpins and a key exonic splicing enhancer is found in the loop of one of the hairpins, which is in turn bound by splicing regulatory proteins such as SRSF1 9The half-life and decay rates of mRNA transcripts in human cells influence protein expression levels. A number of features determine transcript stability including GC content, transcript length, polyA tail length, RBP sites, microRNA binding sites, and mRNA secondary structures Massively parallel reporter assays are high-throughput technologies that enable rapid measurements of thousands of sequences for their regulatory activity and have received widespread adoption in recent years 10Translation can be divided into four phases, initiation, elongation, termination and ribosome recycling Although the vast majority of eukaryotic translation start sites have an AUG codon, often the first AUG codon is bypassed, resulting in usage of more distal AUG codons and alternative protein isoforms. This process is referred to as leaky scanning, with a proportion of ribosomes initiating translation from downstream start codons. Leaky scanning and translational efficiency are influenced by the presence of secondary structures Secondary structures can conceal or expose binding sites for translation regulators, and it has been shown that certain RBPs bind preferentially at structured RNA while others have a preference for linear forms E. coli the lysine riboswitch when lysine is present it restricts translation initiation and also exposes RNase E cleavage sites Riboswitches are components of mRNA molecules that can bind a small molecule and directly control gene expression through RNA conformational changes, without proteins being involved. They are found in both prokaryotes and eukaryotes, with most discovered riboswitches being present in bacteria and archaea BRCA1, a tumor suppressor gene, a longer 5′UTR isoform is expressed only in breast cancer cells, resulting in a 10-fold decrease of translational efficiency due to the formation of a stable complex secondary structure RNA structures can directly interact with the translational machinery and influence the recognition of the translation start 11Early studies indicated that hairpin formation can influence translation efficiency Hairpins at the 5′UTR of ferritin-H and ferritin-L mRNAs act as an iron-responsive element controlling iron levels and are highly dynamic response elements to environmental changes 12RNA G-quadruplexes are enriched at 5′UTRs  where they show a higher frequency at the template strand, suggesting a relative depletion of G-quadruplexes at the RNA level It has also been shown that G-quadruplexes at 5′UTRs of eukaryotic genes can promote translation by favoring recognition of the IRES A study that used massively parallel reporter assays to investigate mRNA translation found that G-quadruplexes in the 5′UTR act as translational inhibitors, and that knockdown of G-quadruplex resolving helicases aggravated these phenotypes 13Regions that are predisposed to secondary structure formation, such as G-quadruplexes have an excess of germline and somatic mutations U2AF1 or SRSF2 mutants, mutations that are commonly observed in myelodysplastic syndrome, results in the accumulation of R-loops MAPT gene, also known as tau, in the interface between exon 10 and intron 10, there is a hairpin structure which can mask the splice site SMN2 and interference with the structure formation is a therapeutic target against the spinal muscular atrophy molecular phenotype Mutations of alternative splicing factors can lead to R-loop accumulation, which may compromise genomic stability and be relevant in the context of cancer pathogenesis TP53 and TERT, where they can modulate splicing and protein isoforms RNA G-quadruplex structures have been identified in several cancer genes, including C9orf72 gene which results in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These repeats form different secondary structures including G-quadruplexes, R-loops and hairpins The expansion of microsatellite repeats at 5′UTRs has been associated with aberrant translation and has been implicated in multiple disorders 14RNA secondary structures are pervasive, interact with RNA binding proteins and are linked to a large number of important functions, including transcription, splicing and translation. Even though the functional importance of secondary structures has been repeatedly demonstrated, the contribution of RNA structures in these processes remains incompletely understood due to the difficulties in identifying dynamic RNA structures and their mechanisms of action. High-throughput technologies enable the systematic investigation of RNA secondary structures and the design of experiments to quantify their contribution in transcription, splicing and translation enables directly testing their mechanisms of action. New methods to dynamically identify RNA secondary structures are gradually revealing their widespread and diverse contributions in gene regulation. However, it remains difficult to capture their dynamic changes across cellular conditions and their interplay with proteins. The degree to which RNA secondary structure formation is influenced by the tissue and cell type remains largely unstudied. The availability of large scale single cell assays will enable the investigation of associations between secondary structures, the presence of various sequence motifs, and expression levels of RBPs across different cell types. Even more interesting could be the combination of single cell technologies with different small molecules that stabilize specific structures.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Non-coding DNA accounts for approximately 98.5% of the human genome. Once labeled as “junk DNA”, this portion of the genome has undergone a progressive re-evaluation and it is now clear that some of its transcriptional products, belonging to the non-coding RNAs (ncRNAs), are key players in cell regulatory networks. A growing body of evidence demonstrates the crucial impact of regulatory ncRNAs on mammalian gene expression. Here, we focus on the defined relationship between chromatin-interacting RNAs, particularly long non-coding RNA (lncRNA), enhancer RNA (eRNA), non-coding natural antisense transcript (ncNAT), and circular RNA (circRNA) and epigenome, a common ground where both protein and RNA species converge to regulate cellular functions. Through several examples, this review provides an overview of the variety of targets, interactors, and mechanisms involved in the RNA-mediated modulation of loci-specific epigenetic states, a fundamental evolutive strategy to orchestrate mammalian gene expression in a timely and reversible manner. We will discuss how RNA-mediated epigenetic regulation impacts development and tissue homeostasis and how its alteration contributes to the onset and progression of many different human diseases, particularly cancer. Since ijection) . The revIn the past 20 years, a new player has emerged as one of the fundamental regulators of this complex scenario: non-coding RNA (ncRNA), which can be divided into short non-coding RNA  and long non-coding RNA . The idea that RNA could act as a structural component of chromatin was proposed already in 1975, when Paul and Duerksen noted how the amount of RNA associated with heterochromatin was twice as much as the one associated with euchromatin . HoweverIndeed, being capable of binding DNA, proteins, and other RNA molecules in a targeted manner, ncRNAs can mediate the interaction of the epigenetic machinery with DNA by sequestering chromatin modifiers by guiding them to the correct genomic location or by serving as molecular scaffolds to coordinate the binding of different interactors . ncRNAs Among the most important mechanisms regulating cell differentiation and embryonic development, the antagonistic action of PRC2 and TrxG/MLL complexes, belonging respectively to the Polycomb (PcG) and Trithorax (TrxG) group proteins, orchestrates the expression of a large number of developmentally regulated genes through controlling the balance between the repressive mark H3K27me3 and the permissive marks H3K4me1/2/3 .Fendrr  was identified as a nuclear-localized transcript, divergently produced from the Foxf1 gene, whose expression is crucial for the caudal lateral plate mesoderm (LPM) development  and Pitx2 (in trans) in vitro, Fendrr plays a fundamental role in controlling H3K27me3/H3K4me3 levels at these genomic sites, therefore regulating the expression of its target genes. Indeed, by forming an RNA-DNA triplex structure, Fendrr can function as a scaffold that promotes the binding of PRC2, thus antagonizing that of TrxG/MLL, inducing H3K27me3 deposition and repressing the target gene (Fendrr loss of function results in decreased SUZ12 and EZH2 (two core components of PRC2) occupancy and increased H3K4me3 levels on the promoter of Foxf1, Pitx2, and Irx3, important genes for the control of lateral plate mesoderm (LPM) development; the resulting altered epigenetic profile is responsible for severe impairments in lateral mesoderm differentiation, in heart and body wall development and, ultimately, in embryonic lethality around E13.75  regulates the activity of TGF-β pathway genes by binding distal GA-rich elements through the formation of RNA-DNA triplexes , that acts via an RNA-DNA triplex to recruit the histone acetyltransferase (HAT) p300/CBP on a poised enhancer, whose transcription promotes SPHK1 expression. Interestingly, the replacement of the triplex-forming region (TFR) of KHPS1 with that of MEG3 is enough to guide KHPS1 on the TGFBR1, a gene normally targeted by MEG3  binds EZH2 with a 524 nt secondary structure similar to that of Fendrr  pathway upon stress or hormonal stimuli and ultimately results in the induction of the genes responsible for heart hypertrophy  phosphorylation (Bvht) and cardiac mesoderm enhancer-associated non-coding RNA (CARMEN). The first one interacts with SUZ12 to regulate the expression of several cardiac precursor cells (CPCs) genes, including the master gene MESP1, potentially by displacing PRC2 from pro-differentiative genes or by recruiting it to repressors of cardiac differentiation program  is of crucial importance in regulating the lifespan of myeloid cells  and juvenile myelomonocytic leukemia (JMML)  .PINC) interacts with RbAp46, SUZ12, and EZH2 through evolutionarily conserved loops in its 5’ region to regulate milk proteins production in alveolar cells of the mouse mammary gland; indeed, the decrease in PINC expression during the transition from late pregnancy to lactation, in which alveolar cells undergo terminal differentiation upon lactogenic hormone stimulation, suggests that this RNA could act as a break preventing milk production and secretion until parturition  and PINT (p53-induced non-coding transcript). GIHCG is aberrantly upregulated in hepatocellular carcinoma (HCC), where it recruits both PRC2 and DNA methyltransferase 1 (DNMT1) on the promoter of miR-200b/a/429, a microRNA (miRNA) often epigenetically dysregulated in cancer  regulates myogenesis through the recruitment of Suv3-9, a histone methyltransferase that catalyzes H3K9me3 deposition, on MyoD promoter , transcribed from the 5′ region of HOXA gene cluster, recruits WDR5/MLL and acts in cis, through chromosomal looping, to coordinate the activation of HOXA 5′ genes  is a trans-acting RNA expressed in T cells that recruits WDR5 on the interferon-γ locus and therefore regulates the inflammatory response upon microbial infection , specifically expressed in patients affected by facioscapulohumeral dystrophy (FSHD) and transcribed from the subtelomeric region 4q35, a region found mutated in 95% of FSHD cases; DBE-T recruits the TrxG protein Ash1L to de-repress 4q35 genes, that in adult cells are normally silenced by H3K27me3 marks, and that in FSHD are aberrantly re-expressed due to a TrxG/PcG epigenetic switch consisting of a loss in H3K27me3 paralleled by an AshL1-mediated H3K36me2 deposition , whose importance in gene regulation has often been debated. eRNAs are short (median 346 nt), unstable, and unspliced nuclear-retained transcripts, bidirectionally transcribed by RNAPII from regions marked with the permissive marks H3K4me1 and H3K27ac . The idepression  through pression , regulatpression  or modulpression .CARMEN  was one of the first lncRNAs to be discovered. It plays a pivotal role in the X-chromosome inactivation (XCI) process, evolved in therian mammals as a mechanism for compensating the dosage of X-related genetic products in XX individuals  on the X-chromosome. The “choice” between the two Xist alleles appears to be random, and the process can be initiated only when Xic is present on at least two chromosomes. Once induced, Xist completely coats the X-chromosome from which it is expressed, causing its progressive inactivation  to exclude RNA polymerase II (RNAPII) from the Xist-coated X-chromosome , a modification that can be recognized by JARID2, that in turn recruits PRC2. The subsequent H3K27me3 deposition serves as a signal for the binding of canonical PRC1, which reinforces the transcriptionally silent state through more H2AK119ub deposition  are present in tandem repeats throughout the genome, transcribed from RNA polymerase I (RNAPI) promoters that in mice are located ∼2 kb upstream rRNA transcription start site . RNAPI bNA genes  , that span from rDNA gene body to intergenic, promoter, and enhancer regions , ranging from 100 bp to 9 kb, that originates from subtelomeric CpG islands that serve as promoters , a histone deacetylase, and its overexpression or knockdown lead to an increase or a diminution, respectively, of H3K9me3, H4K20me3, and hypoacetylated H4   .TERRA can directly bind one of the two telomeric DNA strands, forming a particular secondary structure, termed R-loop, composed of the RNA-DNA hybrid and the unbound DNA strand. Interestingly, it has been proposed that the accumulation of TERRA-induced RNA-DNA hybrids may serve as a trigger for homology-directed repair (HDR), thus promoting alternative lengthening of telomeres (ALT)  . In factnescence . In humaearly G1 , and is early G1 , thus sucis- or trans-acting factors such as transcription factors or lncRNAs such as Kcnq1ot1 and Airn.Genetic imprinting is an intergenerational epigenetic mechanism that ensures the parent-of-origin-dependent expression of one of two alleles. Usually, imprinted genes are organized in clusters, each individually regulated by an imprinting enter, that share common patterns of differentially methylated regions (DMRs) or histone modifications. These modifications are established in the mature germ cells of an individual and last for one generation, being eventually erased in the germline precursor of their offspring . ImprintKcnq1ot1 is a 91 kb RNAPII-transcribed RNA oriented in antisense to the potassium voltage-gated channel subfamily 1 (KCNQ1) gene, located at human chromosome 11p15.5, that is exclusively expressed on the paternal chromosome, as its control region is methylated on the maternal chromosome , regulating various miRNAs availability through sponging mechanisms  gene in an antisense orientation, is imprinted and only expressed from the paternal chromosome, where it silences in cis its target genes Igf2r and the solute carrier family 22 members Slc22a2 and Slc22a3. Specifically, Igf2r is silenced in embryonic, extraembryonic, and adult tissues, whereas the other two genes are only silenced in some extraembryonic tissues , which precludes RNAPII recruitment on its promoter , a highly abundant and heterogenous class of RNAs that was possible to characterize in the last 20 years through the development of powerful techniques such as cap analysis gene expression (CAGE) and single strand RNA sequencing (ssRNA-Seq) .The previously shown lncRNAs RNA-Seq) . NATs arRNA-Seq) . NATs caOne of the hypothesized mechanisms through which ncNATs can regulate gene expression is TI. If widespread, a negative correlation between sense-antisense pairs expression should be expected since TI is thought to be a downregulatory mechanism. Nevertheless, the majority of significant correlations between the expression of the antisense transcripts and that of their corresponding sense transcripts seem to be positively linked . Indeed,HOTAIR, ANRIL, CCAT2, and TUG1, all of which interact with PRC2 subunits , a subunit of the histone demethylase/deacetylase CoREST repressor complex, to guide them on the HOXD locus, thus exerting a dual inhibitory control by simultaneously adding repressive marks and removing permissive ones  regulates PTEN expression both at the transcriptional and post-transcriptional levels. PTENpg1 is transcribed in both senses, giving rise to different ncRNAs: a sense transcript and two antisense RNAs, PTENpg1 asRNA α and PTENpg1 asRNA β. The sense transcript, PTENpg1, acts in the cytoplasm as a sponge for miRNAs that would otherwise target PTEN mRNA, thus increasing its stability. However, it lacks a poly(A) tail, and thus it cannot be efficiently exported from the nucleus. On the other hand, the two antisense RNAs are polyadenylated and exert two different functions: the α isoform guides PRC2 and DNMT3a on PTEN 5’ untranslated region (UTR), located to a different chromosome, to repress its transcription in trans; the β isoform binds PTENpg1, favoring its nuclear export, through the formation of an RNA-RNA duplex whose poly(A) can be recognized by the nuclear transport machinery  structure established by how chromosomes are folded and bound by sub-nuclear structures. In recent years, the development of powerful high-throughput techniques, such as Hi-C, allowing to map physical interactions between chromosome portions, led to the observation that interphasic chromatin is hierarchically organized into high-order structures such as topologically-associated domains (TADs), lamina-associated domains (LADs) and chromatin loops . LADs arThe large amount of RNAs associated with chromatin suggests an important function of these molecules in chromosomal structure maintenance. Indeed, already in 1989, it was evident that a global RNA depletion results in profound nuclear str abnormalities . The abiMALAT1, are diffusely present on chromatin, whereas others form spatially concentrated “hubs” harboring functionally related ncRNAs that often co-localize with the genomic loci from which these RNAs are transcribed. This last category includes transcripts involved in heterochromatin formation, gene expression regulation, and RNA processing, such as Kcnq1ot, that localizes in a TAD encompassing all the imprinted genes and excluding the non-imprinted ones, Airn, Xist, and the small nuclear RNA (snRNA) U7  U7 . NotablynRNA) U7 .In the last couple of decades, small non-coding RNAs (sncRNAs), defined as non-coding transcripts shorter than 200 nt, emerged as critical players in all aspects of gene expression regulation, including epigenetic modulation.Drosophila, favoring the recruitment of chromatin modifiers, promoting the euchromatinization of some specific sub-telomeric heterochromatic regions  are covalently closed RNA molecules that originate from a particular form of splicing termed backsplicing, in which a downstream donor site is linked to an upstream acceptor site. This is often due to physical constraints imposed by the self-recognition of two complementary inverted repeats  flanking the two splicing sites .The absence of free 5′/3′-OH ends makes circRNAs extremely stable molecules, with a half-life (median 18.8–23 h) at least 2.5 times longer than linear transcripts , so stabFECR1, is able to simultaneously recruit TET1 and repress DNMT1 to decrease the methylation levels of the oncogene Friend leukemia virus integration 1 (FLI1), aberrantly overexpressed in various tumors, promoting metastasis in breast cancer , modulates the levels of the PRC1 component BMI1 via miR-802 sponging  complex on the promoter of the pro-inflammatory molecule BATF (B. While they have traditionally been associated with miRNA sponging functions , recent t cancer  (Figure t cancer . circRNAnging Y.  and promnging Y.  (Figure nging Y. . circKcn BATF B.  (Figure  BATF B. .A significant fraction of the genome-wide transcription of non-protein coding RNAs is involved in nuclear regulatory processes. Particularly, ncRNAs have emerged as pivotal players in the epigenetic regulation of gene expression. Indeed, being physically associated with chromatin, various RNA are crucial components of chromatin in eukaryotes as well as critical constituents of ribonucleoprotein complexes involved in the modulation of local and global chromatin states .cis at their site of transcription or recruited in trans to other loci, RNA is potentially able to reach any genomic region and mediate the modulation of its chromatin architecture, resulting in an altered transcriptional readout. Despite the recently developed technologies that advanced our understanding of RNA-mediated modulation of chromatin state, many aspects of this complex field are still debated. For instance, biochemical and molecular details of the RNA-binding specificity of PRC2 and its regulation by the large number of lncRNA interacting with it are still missing (HOTAIR-mediated transcriptional repression (RNA has the unique feature of being able to bind both protein and nucleic acid. Sequence-based and structure-based molecular interactions are promoted by specific sequence motifs as well as by a variety of secondary and high-order structures, respectively. On the other hand, RNA can bind specifically and directly to other nucleic acids as a triple helix or R-loop. Binding flexibility and target specificity make RNA the most suitable molecule to bring functions exerted, for instance, by chromatin modifiers, to specific genomic targets. Indeed, through being either retained in  missing . Additio missing , as suggpression .MALAT1, are diffusely present on chromatin, whereas some others form spatially concentrated “hubs,” harboring functionally related ncRNAs that often co-localize with the genomic loci from which these RNAs are transcribed. This last category includes transcripts involved in heterochromatin formation, gene expression regulation, and RNA processing, such as Kcnq1ot, Airn, and Xist (The large amount of RNAs associated with chromatin suggests an essential function of these molecules also in chromosomal structure maintenance. The ability to interact with both DNA and other RNA molecules, as well as with proteins, makes RNA a versatile scaffold, efficiently serving as a bridge to coordinate the assembly of various nuclear compartments. Recently, the development of RD-SPRITE provided important experimental proof of the mechanisms through which RNA shapes chromatin architecture . The datand Xist .The first evidence of the importance of RNA in maintaining nuclear structure and activity was provided in 1989 . Nowaday


Recombination and mutation of viral genomes represent major mechanisms for viral evolution and, in many cases, moderate pathogenicity. Segmented genome viruses frequently undergo reassortment of the genome via multiple infection of host organisms, with influenza and reoviruses being well-known examples. Specifically, major genomic shifts mediated by reassortment are responsible for radical changes in the influenza antigenic determinants that can result in pandemics requiring rapid preventative responses by vaccine modifications. In contrast, smaller mutational changes brought about by the error-prone viral RNA polymerases that, for the most part, lack a replication base mispairing editing function produce small mutational changes in the RNA genome during replication. Referring again to the influenza example, the accumulated mutations—known as drift—require yearly vaccine updating and rapid worldwide distribution of each new formulation. Coronaviruses with a large positive-sense RNA genome have long been known to undergo intramolecular recombination likely mediated by copy choice of the RNA template by the viral RNA polymerase in addition to the polymerase-based mutations. The current SARS-CoV-2 origin debate underscores the importance of understanding the plasticity of viral genomes, particularly the mechanisms responsible for intramolecular recombination. This review describes the use of the cystovirus bacteriophage as an experimental model for recombination studies in a controlled manner, resulting in the development of a model for intramolecular RNA genome alterations. The review relates the sequence of experimental studies from the laboratory of Leonard Mindich, PhD at the Public Health Research Institute—then in New York City—and covers a period of approximately 12 years. Hence, this is a historical scientific review of research that has the greatest relevance to current studies of emerging RNA virus pathogens. Homologous and heterologous recombination in positive-strand RNA viruses, particularly poliovirus and coronavirus, has been identified and studied ,7,8,9,10In light of the emergence of the COVID-19 pandemic caused by the human coronavirus pathogen SARS-CoV-2, the lesson provided by the cystovirus-based discoveries must be revisited. Although hardly evident 20 years ago, the subject of RNA genome reorganization has taken center stage, as the origin of the SARS-CoV-2 virus is a recognized result of genome recombination among mammalian coronaviruses . It mustCystoviridae)  are a unique group of viruses that have proven of great utility in the study of many facets of molecular virology . O. O25]. OP. syringae pv. phaseolicola HBMPO.16 spheroplasts which were then plated on a lawn of intact P. syringae pv. phaseolicola HB10Y bacterial cells. Selected plaques were confirmed as the rescue noted by the small deletions inserted into the viral genome. Despite a modest beginning, the procedure shortly demonstrated its utility for the incorporation of foreign genes into the viral genome. Onodera et al. (1992) exploited the technique by inserting a transcript containing the kanamycin resistance gene (kan) into a viable virus and established a carrier state (CS) in the host cells . Th. ThfunctTo conclude, it stands to reason that the ultimate understanding of viral genomic RNA recombination mechanisms will be dependent on the entire delineation of the molecular architecture and geometry of the RNA as it enters and packages within the mature virion particle. The description of the cystovirus genome as a liquid crystalline array in a highly compacted form represents a start in order to complete the goal , and sig


Caenorhabditis elegans. We measured how degrading and re-expressing orthologs of EBF3, BRN3A and DYNC1H1 at multiple periods throughout development affect 30 morphological, locomotor, sensory and learning phenotypes. We found that phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of rescue varied by gene and phenotype. This work highlights the critical need to assess multiple windows of degradation and re-expression and a large number of phenotypes to understand the many roles a gene can have across the lifespan. This work also demonstrates the benefits of using a high-throughput model system to prioritize NDD risk genes for re-expression studies in other organisms.Recent studies have indicated that some phenotypes caused by decreased function of select neurodevelopmental disorder (NDD) risk genes can be reversed by restoring gene function in adulthood. However, few of the hundreds of risk genes have been assessed for adult phenotypic reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of NDD risk gene orthologs using a conditional protein degradation system and machine-vision phenotypic profiling in  Summary: We identified diverse temporal functional windows and phenotypic reversibility profiles for three neurodevelopmental disorder risk genes: EBF3•unc-3, BRN3A•unc-86 and DYNC1H1•dhc-1. Re-expression of certain genes could rescue multiple phenotypes later in life, prioritizing them for study. Neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID) are highly genetically heterogeneous and are accompanied by a range of cognitive and behavioral phenotypes including sensory processing and learning impairments . More seHistorically, it was assumed that any treatment targeting neurodevelopmental disorder risk genes would need to be administered very early in development to be effective. This long-held assumption was challenged by reports that re-expression of several risk gene orthologs could reverse multiple altered neurophysiological and/or behavioral phenotypes in adult mice . In addiThe handful of reports that show the possibility of phenotypic reversibility with gene reactivation in adult mice offer critical insights into which genes are promising candidates for future re-expression-based therapies. However, because of limitations including cost, developmental rate and technical difficulties , very few risk genes have been tested for adult phenotypic reversibility. Further, the rapidly growing number of risk genes identified in recent years has exacerbated this problem and drastically increased the need for candidate prioritization to better direct research efforts. While most neurodevelopmental disorder risk genes are highly expressed early in pre-natal development , many coCaenorhabditis elegans offers multiple advantages to systematically assess the temporal requirements and phenotypic reversibility of neurodevelopmental disorder risk genes. There are C. elegans orthologs for a high number of neurodevelopmental risk genes  were seE. coli OP50 liquid culture 48 h before use. All plates were stored in the dark at room temperature (20°C) in a temperature and humidity-controlled room  per strain. The mean of each condition was then compared using a two-tailed unpaired t-test and Benjamini–Hochberg control of false discovery rate at 0.01. Figures were generated using the ggplot2 and complex heatmap packages in R (We used the MWT software (version 1.2.0.2) to deliver mechanosensory stimuli and acquire images , and Choges in R .Degradation groups were created as described above. All images were taken using 96-h-old animals. Adult animals were anesthetized by being placed in 10 µl of 7.5 mM Levamisole (Sigma-Aldrich) and 0.225 M BDM  (Sigma-Aldrich) on glass microscope slide containing a 2% agar pad. After animals were paralyzed, a 1.5 coverslip was placed on top the agar pad. A Leica SP8 white light laser confocal microscope and 63× oil immersion lens was used for imaging. Step size was 0.3 µm. GFP was excited using a 488 nm wavelength laser with emitted light collected through a 490-778 nm bandpass filter. mNeonGreen was excited using a 506 nm wavelength laser with emitted light collected through a 512-742 nm bandpass filter. For each condition, four to five animals were scored for the presence of GFP or mNeonGreen. Final figures were generated using ImageJ .ieSi57[eft-3p::TIR1::mRuby::unc-54 3'UTR+cbr-unc-119(+)] II; OH13988 ieSi57[eft-3p::TIR1::mRuby::unc-54 3'UTR+cbr-unc-119(+)] II; unc-3(ot837[unc-3::mNeonGreen::AID]) X; OH15227 unc-86(ot893[unc-86::3xFlag::mNeonGreen::AID]) III; CA1207 dhc-1(ie28[dhc-1::degron::GFP]) I; CA1210 ieSi57[eft-3p::TIR1::mRuby::unc-54 3'UTR+cbr-unc-119(+)] II; dhc-1(ie28[dhc-1::degron::GFP]) I.The following strains used are available through the CGC: CA1200 ieSi57[eft-3p::TIR1::mRuby::unc-54 3'UTR+cbr-unc-119(+)] II; unc-86(ot893[unc-86::3xFlag::mNeonGreen::AID]) III; VG946 mizSi6 V; dhc-1(ie28[dhc-1::degron::GFP]) I.The following strains were generated using standard genetic crosses: VG937 TIR1 sequence: forward, 5′-GACCGTAACTCCGTCTCC-3′; reverse, 5′-CGTTGGTGGTGATGATTTGAC-3′; AID degron sequence: forward, 5′-CCTAAAGATCCAGCCAAACC-3′; reverse, 5′-CTTCACGAACGCCGC-3′; or forward, 5′-GATCCAGCCAAACCTCCGGC-3′; reverse, 5′-CTTCACGAACGCCGCCGC-3′.Successful crosses were determined through visual confirmation of fluorescent reporters as well as PCR-based genotyping using the following primers:


In recent years, cancer immunotherapy has made remarkable achievements. Immune checkpoint inhibitors (ICIs) have been used successfully in several types of cancer in the past decade. However, expanded indication and increased use of Immune checkpoint inhibitors have resulted in increased reports of toxicity called immune-related adverse events (irAEs). Due to the unique immunological characteristics of the liver, a hepatic immune-related adverse events has also been reported, which is usually termed Immune-mediated hepatitis (IMH). So far, it is generally considered that the mechanism of IMH induced by Immune checkpoint inhibitors is mainly the overactivation of T cells. It has been reported that the incidence of IMH ranges from 1% to 15%. Because of the lack of specific markers, a diagnosis of exclusion of IMH is critical. Although most IMH is mild and recoverable, several death cases have been reported, which has been increasingly concerned. This review summarizes the current understanding of the pathophysiology, epidemiology, diagnosis, management and prognosis of IMH caused by Immune checkpoint inhibitors. It also discusses the controversial issues in IMH, such as the role of liver biopsy, grading criteria, risk factors, rational treatment strategies with steroids, and the timing of Immune checkpoint inhibitors rechallenging, which may provide helpful information for IMH in future clinical practice. In the past decade, immune checkpoint inhibitors (ICIs) have developed rapidly in the application of advanced malignancies . AccordiHowever, with the wide application of ICIs, several unexpected immunological and inflammatory events, termed immune-related adverse events (irAEs), have been reported . It has This review aims to discuss the pathophysiology, epidemiology, diagnosis, management and prognosis of IMH caused by ICIs and provide references for the clinical application of ICIs.The critical step for ICIs in cancer immunotherapy is the activation of T cells. As mentioned above, CTLA-4, PD-1 and PD-L1 are three current ICIs targets. However, the mechanisms of these ICIs are different. It has been demonstrated that the CTLA-4 inhibitors play a role in the initial phase, while PD-1 and PD-L1 inhibitors are involved in the effector phase . In the + Treg cells, protect the liver from autoimmune responses to antigens by downregulating effector T cells  production, which can go on to activate cytotoxic T lymphocytes , as wellm of IMH . ICIs coantigens .+ T cells in the systemic circulation with Kuffer and LSECs, leading to the retention of activated CD8+ T cells in the liver  express the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). These continuously expressed adhesion molecules and the slow blood flow in the hepatic sinusoids promote the interaction of activated CD8he liver . Upon rehe liver , which whe liver , leadingThe incidence of IMH is mainly counted through the reports of irAEs in clinical trials. Up to date, most trials defined the occurrence and grades of irAEs based on the common criteria for adverse events (CTCAE), which was used by referring to the elevations of aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin based on the upper limit of normal (ULN).The reported incidence of IMH varies according to the different agents, dosages and indications . It has In addition, it is worth noting that there are some commonalities between other irAEs and IMH. The incidence of other irAEs was also associated with different treatment strategies. It has been reported that the incidence of rash, colitis and diarrhea is higher in patients treated with anti-CTLA-4 than in patients treated with PD-1  . The risAlthough IMH occurs less commonly than some other non-hepatic irAEs, fatal cases have been observed in both clinical trials and post marketing phase. A meta-analysis investigated the fatality rates caused by ICIs, which indicated that in 613 reported fatal cases, 124 were secondary to IMH. Furthermore, in this study, of all fatal cases, 31 (5.1%) in the ipilimumab group, 74 (12.1%) in the anti-PD-1/PD-L1 group and 19 (3.1%) in the combined therapy group were caused by IMH. The study further analyzed the patients with melanoma from seven international academic medical centers and found that 21 fatal cases were reported, of which 5 (23.8%) cases were caused by IMH, followed by myocarditis (28.6%) and colitis/enteritis (28.6%) . These sAlthough certain risk factors have been associated with irAEs during ICIs therapy, the risk factors associated with IMH have not been fully elucidated . There aFrom the perspective of the treatment strategy of ICIs, it has been reported that the incidence of irAEs in monotherapy of anti-CTLA-4 is higher than that in anti-PD-1 or PD-L1, which suggests that the types of ICIs may be a risk factor in IMH. Furthermore, the risk of incidence of IMH is correlated with the dosage of ICIs. In a study of ipilimumab for melanoma, serious hepatic adverse events were more common at 10 mg/kg compared to the dosage of 3 mg/kg (30% vs. 0%) . AdditioAs for the drugs other than ICIs, it has been reported that acetaminophen was associated with a 2.1-fold increased risk of all grades of IMH and the use of 3-hydroxy-3-methyl-glutaryl-coenzyme reductase inhibitors was associated with a 4.7-fold increased risk of grade 3 or higher IMH compared with untreated .p = 0.003). Furthermore, several studies have demonstrated that patients with autoimmune disorders such as thyroiditis or rheumatological have a higher risk of IMH during ICIs therapy  was an independent risk factor for IMH (p < 0.05) was another independent risk factor for IMH   , which s= 0.002) , which sp < 0.001)  . In sum Although most cases of IMH are asymptomatic, a few patients may present with fatigue, abdominal discomfort, fever, rash, and rarely jaundice . Acute lAbnormal elevations of serum liver enzymes in liver function tests are usually indexed in the diagnosis of IMH. Elevations of ALT or AST more than two times ULN should be concerned. Sometimes it should also be concerned mild to moderate elevation of serum ALP >2.5 × ULN, and abnormal elevation of total bilirubin >1.5 × ULN. Since the IMH is usually asymptomatic or has non-specific symptoms, many cases are diagnosed during monitoring during ICI therapy. Furthermore, specific biomarkers of IMH have not been elucidated. Although recent studies have demonstrated that human leukocyte antigen and IL-6 are susceptible to liver injury induced by ICIs, there is no specificity in IMH, which needs more studies to verify . For timThe 2019 European Association for the Study of the Liver (EASL) guideline for drug-induced liver injury (DILI) classified IMH as a special type of DILI . SimilarThe differential diagnosis of IMH is still challenging as the existing a lot etiologies of abnormally elevated liver enzymes, which mainly include drugs other than ICIs, viral infection, autoimmune and metabolic diseases, tumor-related causes, biliary diseases, musculoskeletal and cardiovascular system diseases . TherefoIdentification of the above differential causes of liver injury during ICIs therapy requires a detailed medication history. It is noteworthy that chemotherapeutic drugs combined with ICIs, such as dacarbazine, carboplatin, and bevacizumab, which may also cause liver injury during cancer immunotherapy . Further+ T cells and eosinophils, less frequently CD4+ T cells, B cells, and plasma cells , are clinically used to evaluate IMH . Both grRecently, American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), Society for Immunotherapy of Cancer (SITC), National Comprehensive Cancer Network (NCCN), and EASL have developed guidelines on irAEs, including IMH, to guide the management of irAEs . Due to Most guidelines recommend that before every ICIs administration, all patients should check liver parameters, especially for patients with a background in chronic viral hepatitis, which is recommended an antiviral therapy before the first time of ICIs therapy. To patients with asymptomatic elevations of liver tests and excluded other suspicious causes, IMH induced by ICIs should be considered. Unlike other DILI, it is not enough to discontinue the suspected culprit drugs in the management of IMH as IMH is usually induced by excessive immune response of the liver, so initiation of immunosuppressive therapy is equally necessary. Currently, all recommended management of IMH suggests using corticosteroids such as prednisone, methylprednisone, or equivalent . FurtherAlthough guidelines recommend using a dosage of prednisone from 0.5 to 1 mg/kg/day for grade 2 IMH and initiating methylprednisone 1–2 mg/kg/day in more severe IMH, the timing for corticosteroid administration is still controversial. It has been shown that nearly half of patients with grade 3 or 4 IMH who discontinue ICIs can improve spontaneously without a corticosteroid treatment . AnotherCurrently, most society guidelines recommend corticosteroids as a first-line treatment for IMH. However, some cases of refractoriness on steroids were reported to not respond to steroids or failure to normalize liver function. It was recommended that if there is no response with intravenous methylprednisolone, second-line treatment of 500–1000 mg of MMF twice daily can be considered. In addition to MMF, ASCO also proposed that azathioprine (AZA) can be used as the second-line agent for steroid-refractory IMH after ruling out the infectious causes , and tesvia the proliferator-activated receptor-α-nuclear factor-κB signal pathway . However, according to a systemic review, grade 3 or 4 IMH should be considered to resume ICIs therapy or switch CTLA-4 inhibitors to PD-1/L1 inhibitors when hepatitis severity improves to grade 2 . FurtherMost patients with IMH can recover spontaneously or after corticosteroid administration. For the recovery time, it has been reported that IMH usually resolves in 5–9 weeks . HoweverAs to the oncology outcomes, fewer studies have focused on the clinical outcomes of IMH compared with other irAEs. Despite this, a study showed an excellent outcome and overall survival in patients with IMH , which iWith the success of ICIs in several types of cancer, more and more patients are being treated with ICIs. However, ICIs therapy also causes a variety of irAEs. Due to the immunological characteristics of the liver, ICIs also cause liver-related adverse events, usually termed “immune-mediated hepatitis”. Although IMH is not common compared with other irAEs, with the expanded indications of ICIs therapy, an increasing number of cases diagnosed with IMH are reported. IMH has become increasingly concerned about its potential influence on anti-tumor therapy and lethality. However, the diagnosis and management for IMH are based on found in retrospective case series experience, so there is an urgent need for some randomized clinical trials to clarify the current debate in the IMH, such as further exploring the molecular mechanisms and identifying the prediction markers of IMH as well as evaluate the role of liver biopsy in IMH causality and grading assessment. In addition, we need prospective studies investigating steroid and non-steroid based management of IMH to determine the ideal treatment regimen and better delineate the threshold for appropriate treatment rechallenge after initial management.


Little is known about how easy it is for children to deal with coronavirus-related information, or to what degree children have accurate knowledge about the virus. However, both might influence children’s preventive behaviors. Thus, this study explores children’s coronavirus-related health literacy (CHL) and related knowledge.In a classroom survey among fourth graders’ in North-Rhine Westphalia, Germany (07/21 - 11/21), 4 items were used to assess CHL. 5 items were used to measure coronavirus knowledge, while also assessing the frequency of speaking about the coronavirus with parents. Sociodemographic characteristics were recorded, including family affluence (FAS). Bivariate correlations are reported.n = 364 students are included , the mean age is 9.5 years (SD=.69). 47.5% of the sample find it rather difficult or very difficult to find information about the coronavirus, while this share is 36.1% for understanding, and 39.1% for appraising coronavirus information. 22.4% say it’s difficult to not infect themselves or others (applying information). Dealing with coronavirus-related information is more difficult for children who less frequently talk to their parents about the virus  and have lower family affluence . While certain misbeliefs are rare , the belief that eating healthy prevents an infection (15.6%), that all infected people have coughs and fever (62.8%), and that a face mask doesn't help prevent an infection (44.3%) are more prevalent. Children who have a home language other than German state more inaccurate knowledge (p<.05).This study suggests that children’s needs for accessible and understandable information on the coronavirus were met only party. Also, certain misbeliefs are prevalent which might undermine adherence to preventive measures. More research is needed to verify these findings.• In our sample, children’s needs for accessible and understandable information on the coronavirus were met only party.• Misbeliefs about the coronavirus are prevalent which might undermine preventive measures.


Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA’s structure and its interactions with proteins in the translation process. In particular, the 5′-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5′-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5′-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5′-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.A mouse model has often been used in studies of  ThiTP53 is regulated at several levels, and several proteins are involved in this regulation. Protein factors that bind to p53 mRNA are potentially involved in translation regulation. Regulation at the level of p53 mRNA translation determines the amounts of synthetized p53 protein and its isoforms  calculated as the Fourier transform of the scattering curve. The obtained values were 3.87 and 4.10 nm, respectively. The maximum diameter in the particle Dmax estimated from the pair distribution function was 16.21 nm. The spatial RNA model predicted by the RNAComposer program fits well to the experimental data (χ2 = 1.9) and the theoretical and experimental RNA volumes, which were 19,051 and 21,908 nm3, respectively. Moreover, the SAXS-derived ab initio envelope and the theoretical RNA model have very similar L-shaped structures -derived Rg values  and 1.5 nm (P(r)), and Dmax was 5.07, whereas theoretical data yielded Rg of 1.27 nm and Dmax of 4.17 nm. In this case, we also observed a very good fit of the model. Comparison of the ab initio envelopes of RNAs: -51/9, -38/-2 and -30/-10 reveals that although the initial, longest fragment was shortened by 23 and 39 nucleotides, this did not lead to significant structural rearrangements of its sub-fragments  and C(-2), and a shorter, 21-nucleotide-long RNA oligomer corresponding to the region between U(-30) and G(-10) A,B. Analg values . The calragments . Thus, iNext, we focused on a highly evolutionarily conserved mRNA region spanning nucleotides A47 and U140 . This reWe have earlier observed that in mouse p53 mRNA the presence of an Mdm2-binding hairpin is not indisputable, since four nucleotides, C45, G51, C82 and C83, are different than those present at the corresponding positions in human p53 mRNA, nucleotides U, A, G and A, respectively . This cag values for this RNA were 2.099 nm (Guinier) and 2.078 nm (P(r)), and its Dmax was 7.1 nm -derived Rg values, and the calculated Dmax value was 17.65  spectroscopy to determine the impact of Mgon codon . Structu2+ ions. In the same conditions, RNA-106/-78 showed four positive maxima at 273, 253, 225 and 210 nm and negative maxima at 216, 234 and 307 nm. CD spectra collected for both these RNAs in the buffer with no Mg2+ ions added were characterized by a smaller number of peaks of smaller intensity. In both buffer conditions (with and without Mg2+ ions), an increasing temperature caused decay of weak peaks, and a decrease in the intensity of all the peaks was additionally observed. The results of CD spectroscopy showed that Mg2+ ions stabilize the structure of the studied RNA, causing structural reorientation of their polynucleotide chains.The CD spectra of the analyzed p53 fragments had a broad positive peak covering the region from 260 to 300 nm, along with negative peaks at about 210 nm and about 240 nm . It has The next analyzed p53 mRNA fragment, RNA-122/201, was 323 nucleotides in length, and it spanned the whole 122-nucleotide-long 5′ non-coding region and a 201-nucleotide-long stretch of the coding sequence . It has g of 9.78 and 9.84 nm for Guinier and P(r) analysis, respectively /C(-57) has earlier been described as a binding site for the hnRNP Q protein ,52: our  methods . PossiblIn the case of domain U(-50)/A8, the aforementioned bottom-left part of that domain and the region C(-5):C(-1), immediately preceding the AUG1 initiation codon, are accessible to oligomer hybridization. In both regions, structural distortions are present, which may facilitate oligomer binding. Importantly, the high accessibility of these regions may be beneficial to the binding factors crucial for the regulation of the translation initiation process, as it may regulate the accessibility of the AUG1 codon to the translation machinery. There is also an accessible nucleotide C(-19) in the apical loop of the hairpin motif.The region U35:C46 adjacent to the Mdm2-binding hairpin A46/U81 on its 5′ side; the regions A50:C59 and C79:A86, spanning bulged nucleotides, a U-track; and three bottom-right nucleotides 79-CCU-81 of the hairpin, are also highly accessible to oligomer hybridization. In the case of hairpin A89/U140, the region C94:C108, mostly bulging out from the hairpin, and the region A116:U122 in the apical loop, namely, the regions between which the AUG2 translation initiation codon is located, are accessible to oligomer hybridization. It is important to note that AUG2 is located between regions accessible to hybridization; therefore, it may serve as a facilitator of AUG2 accession for the factors regulating Δ41p53 synthesis.When comparing the accessibility of the 5′-terminal region of mouse p53 mRNA to its counterpart in human mRNA , we can As a next step in our study, we decided to identify proteins which are able to interact with the 5′-terminal region of mouse p53 mRNA. To this end, we used RNA-centric affinity chromatography combined with mass spectrometry analysis ,37,38. IThe mass spectrometry analysis yielded a list of proteins which are potentially able to interact with the 5′-terminal region of mouse p53 mRNA . We focuIn the group of candidates, we identified a set of proteins present in the top 20 positions in the candidates’ group in a similar RNA-centric affinity chromatography experiment that was performed for human cells . We seleFrom the group of protein candidates with high likelihood of interacting with mouse p53 mRNA  and withThe hnRNP K and PCBP2 proteins are highly conserved. A comparison of hnRNP K protein sequences between mouse and human revealed its 100% identity, and in the case of PCBP2, the sequences differ by only 1.3% (5 out of 365 amino acids).d)  was used. Radiolabeled RNA-122/201 was incubated with hnRNP K or PCBP2 proteins, under three different conditions, and in all cases shifts of the bands were observed on the polyacrylamide gels, which indicated the formation of protein–RNA complexes (data not shown). Afterwards, the EMSA experiments were carried out with an increasing concentration of hnRNP K or PCBP2 in order to estimate the dissociation constant values (Kd) A,B. A sieference ; D. M. Jeference ; Figure d value of 95 ± 6 nM reported for the human PCBP2–RNA-P1Δ40p53 complex is higher than Kd of 15 ± 3 nM, which was determined for the murine PCBP2–RNA-122/201 complex. It has earlier been described that PCBP2 binding sites in the human 5′-terminal region of p53 mRNA are distributed along this region -labeled primers: Rm508 (5′-TTCGGAGAAGCGTGACACCCTGC-3′), Rm591 (5′-CCATAAGCCTGAAAATGTCTCCT-3′) and Rm679 (5′-TTCAAAAAACT CCTCAACATCCT-3′) and 50 units of Super Script IV reverse transcriptase . The reverse transcription products were separated on 8% polyacrylamide gels and visualized with the FLA 5100 image analyzer .In order to visualize RNase H cleavages, primer extension analysis was carried out using the following 5′-end- ATP at the 5′ end using T4 polynucleotide kinase, following the standard procedure. Then, the RNA was denatured at 65 °C for 5 min and cooled on ice. The RNA–protein binding reaction was conducted in a buffer containing 25 mM NaH2PO4 pH 8.0, 50 mM NaCl, 25 mM KCl, 2.5 mM MgCl2, 10 mM imidazol, 0.5 mM DTT, 0.5 mM PMSF, 7% glycerol, 0.5 U/μL RNasin, 75 ng/μL BSA and 100 ng/μL yeast tRNA for PCBP2 protein; and 150 mM NaCl, 25 mM KCl, 50 mM Tris-HCl pH 8.0, 5 mM HEPES-KOH pH 8.0, 10 mM imidazole, 5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 75 ng/µL BSA, 100 ng/µL yeast tRNA, 7% glycerol and 0.5 U/μL RNasin for hnRNP K protein. In order to measure the Kd values, [32P]-labeled RNA-122/201 was incubated for 25 min at 4 °C with the protein at the following concentrations: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 nM for PCBP2; and 25, 50, 100, 200, 400, 800, 1600, 2400 and 3200 nM for hnRNP K. Samples were loaded on 4% polyacrylamide gels . Electrophoresis was conducted for 3 h at 4 °C, the gel was dried and the results were visualized using FLA 5100 image analyser (Fuji).Before protein–RNA complex formation, RNA-122/201 was radiolabeled with γ-[


Hepatocellular carcinoma (HCC) is a highly mortal type of primary liver cancer. Abnormal epigenetic modifications are present in HCC, and RNA modification is dynamic and reversible and is a key post-transcriptional regulator. With the in-depth study of post-transcriptional modifications, RNA modifications are aberrantly expressed in human cancers. Moreover, the regulators of RNA modifications can be used as potential targets for cancer therapy. In RNA modifications, N6-methyladenosine (m6A), N7-methylguanosine (m7G), and 5-methylcytosine (m5C) and their regulators have important regulatory roles in HCC progression and represent potential novel biomarkers for the confirmation of diagnosis and treatment of HCC. This review focuses on RNA modifications in HCC and the roles and mechanisms of m6A, m7G, m5C, N1-methyladenosine (m1A), N3-methylcytosine (m3C), and pseudouridine (ψ) on its development and maintenance. The potential therapeutic strategies of RNA modifications are elaborated for HCC. Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide . As an iEpigenetics is a branch in the field of genetics which explores the phenotypes that can be inherited from chromosomal changes which do not modify the nucleotide sequence of genes, of which RNA modification has been a recent hotspot of epigenetic research . In natuThe epigenomic environment is highly complex. RNA modification is an important epigenetic regulatory mechanism, regulating the structure, function, and stability of RNA and controlling the expression of related genes . RNA modFirst discovered in 1974, m6A is the most popular RNA modification . m6A mod6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq) are simple and efficient  and m6A-level and isoform-characterization sequencing (m6A-LAIC-seq), enabling measurement of transcriptome-wide m6A modification levels . The digfficient . Ligatiofficient . Deaminafficient . In addifficient . Howeverfficient . The detThe m6A methylation modification is written by methyltransferase 3 (METTL3), 14 (METTL14), and 16 (METTL16), RNA binding motif proteins 15 (RBM15) and 15B (RBM15B), KIAA1429, and Wilms tumor 1-associated protein (WTAP). It is subsequently erased by the demethylases alkane hydroxylase homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO). Proteins that read the modification include members of the YT521-B homolog (YTH) structural domain family of m6A RNA binding proteins , insulin-like growth factor 2 mRNA-binding protein family , and heterogeneous nuclear ribonucleoprotein family , and proline-rich and coiled-coil protein 2A (PRRC2A) . m6A metThe addition of m6A onto mRNA, long intergenic non-coding RNA (lincRNA), and miRNA is catalyzed by the METTL3-METTL14 complex . METTL3 The addition of m6A is reversible with reliance on a coordinated and highly dynamic system of methyltransferases and demethylases. m6A demethylases FTO and ALKBH5 are part of the non-heme Fe(II)/α-ketoglutarate-dependent dioxygenase family . FTO is m6A has a major role in cell differentiation with tissue development. It can regulate the entire RNA lifecycle, in terms of RNA processing, decay, and translation, through the identification of specific binding proteins, dynamically regulating physiological and pathological processes such as tumorigenesis . m6A wrim6A promotes the binding of regulatory proteins by altering the structure of RNA to regulate gene expression and RNA maturation . The IGFThere is growing evidence suggesting that abnormal changes in the levels of total m6A and its regulators in HCC tissues are strongly connected with poor clinical survival . m6A regvia bioinformatics analysis  as a prognostic factor, promoting HCC cell proliferation, colony formation, migration, and tumorigenicity in vitro   pathway involvement in HCC progression . In addiSorafenib is a first-line drug approved for treating advanced HCC. However, sorafenib resistance reduces its efficacy in HCC. METTL3 via METTL3, and is an important oncogene in HCC progression, providing a new therapeutic target for HCC   . TransarGNA O 1) . A studypatients .ALKBH5 expression is downregulated in HCC and inhibits the proliferation as well as the invasive ability in HCC cells, which is an indicator of an independent prognosis in HCC patients. ALKBH5-mediated m6A demethylation in HCC cells attenuates LY6/PLAUR domain containing 1 (LYPD1) expression and inhibits the malignant progression of HCC  expression to regulate the WNT/β-catenin axis to exert oncogenic function as a potential HCC therapeutic strategy  and 14 (ATG14) in an m6A-dependent manner to drive hypoxia-induced HCC autophagy It was shown that radiofrequency ablation (IRFA)-deficient sublethal heat stress promotes HCC progression by upregulating EGFR expression levels through an N6-methyladenosine mRNA methylation-dependent mechanism  expression and promoting HCC progression  expression in an m6A-dependent manner, promoting HCC progression . miRNA 1CC cells . YTHDF2 CC cells . It was CC cells . It was gression .GLI1) mRNA through interaction with IGF2BP1 , WD repeats structural domain 4 (WDR4), and Williams-Beuren syndrome chromosome region 22 protein  . m7G modMETTL1 is shown to be upregulated in HCC and promotes HCC migration and proliferation through the phosphatase and tensin homolog (PTEN) as well as the AKT signaling pathway (WDR4 expression significantly increased m7G methylation levels in HCC and conferred a poor prognosis. WDR4 can enhance HCC progression by promoting cyclin B1 (CCNB1) mRNA stability and translation and is a candidate HCC therapeutic target  is mainly distributed in archaea, prokaryotes, and eukaryotes, but is only conserved in revisiae   were related to poor prognosis in HCC patients  and promoting the binding of H19 to G3BP stress granule assembly factor 1 (G3BP1) (NSUN2 overexpression increased the stability of Fizzy-related-1 (FZR1) mRNA and promoted the proliferation of HCC cells  . AnotherCC cells . These fFirst discovered in 1961, m1A is widely distributed in bacteria, archaea, and eukaryotes and is evolutionarily conserved  Dunn, 1. m1A is m1A sequencing has enabled a comprehensive exploration of m1A methylation modifications in the transcriptome, providing an important tool for exploring m1A methylation function. m1A blocks Watson-Crick pairing, effectively blocking RT and inducing truncation or mutation of RT products . Based oTRMT6 and TRMT61 reduces proliferation and death of C6 glioma cells, while their overexpression biological processes encode mRNAs involved in tumorigenic processes , readers  and demethylases  . TRMT6 arocesses . The m1Arocesses . m1A58 mfication . ALKB prfication . The YTHfication . YTDHF pYTHDF1, YTHDF2, YTHDF3, and ALKBH3 expression levels were upregulated in HCC using a raw letter analysis and were closely associated with HCC metabolism, providing new insights into m1A modification and metabolic heterogeneity in HCC 、AlkAniline-seq and Hydrazine-aniline cleavage sequencing (HAC-seq) . These mALKBH3 is associated with tumor development and modification of protein synthesis, indicating that m3C may play a prominent part in cancer biology , 2B (METTL2B), 6 (METTL6), and 8 (METTL8), all contain conserved S-adenosylmethionine (SAM) binding domains. ALKBH1 and ALKBH3 demethylate m3C in tRNAs, affecting RNA stability and preventing degradation . METTL2  biology .METTL6 mRNA expression levels were significantly upregulated in HCC and were closely associated with poorer patient survival outcomes. In contrast, METTL6 downregulation inhibited HCC progression by suppressing cell adhesion molecules and could be a potential therapeutic target for HCC , telomerase RNA, lncRNA, and polyadenylated mRNA , 2 Cohn. When boAs research has progressed, several methods for Ψ have been developed. Mainly include Ψ-seq、PSI-seq、Pseudo-seq、CeU-Seq、HydraPsi-Seq and Nanopore RNA sequencing . These mΨ is the most abundant type of post-transcriptional modification in ncRNA. An essential difference between pseudouridylation and methylation is that pseudouridylation occurs as an irreversible modification in mammals . The forDKC1 can serve as a marker of the proliferative potential of HCC cells and is an unfavorable prognostic factor for HCC (Ψ was the first RNA modification shown to be associated with cancer, and Ψ modifications and regulators have been proved to take an important part in HCC  Zhao et. The stu for HCC . One stu for HCC . Except  for HCC . PUDP po for HCC . These sCurrently, aberrant post-transcriptional modifications in cancer cells are gradually being studied, and there are many assays to detect aberrant RNA epigenetic modifications in tumors . Studiesin situ into the left liver lobe of nude mice using in situ transplantation experiments, and found that knocking out METTL3 significantly inhibited the growth of HCC tumors in situ in the liver of nude mice (With the advancement of gene editing technology, gene knockdown of tumor-related genes using gene editing systems such as CRISPR/Cas9 has been widely studied . Currentude mice . Anotherude mice . In the ude mice . These aRNA modifications play a key role in regulating cell fate, an important regulator of HCC, in tumorigenesis and disease progression . The m6AIn conclusion, although RNA modifications and differential expression of associated regulatory genes have been explored in most physiological processes of HCC and development, their precise role and effects on tumorigenesis, proliferation, metastasis, and resistance still require further investigation. In this review, we have summarized existing knowledge on the important roles and regulatory mechanisms of m6A, m7G, m5C, m1A, m3C, and ψ in HCC, suggesting that targeting aberrant post-transcriptional modifications in cancer cells has the potential to be an effective tool for HCC treatment.


Acropora formosa) between the highly variable reef flat and the less variable reef slope environments. Coral colonies originating from the reef flat had higher rates of survival and amassed greater protein densities but calcified at reduced rates compared to conspecifics originating from the reef slope. The energetics of both populations however potentially benefited from greater light intensity present in the shallows. Reef flat origin corals moved to the lower light intensity of the reef slope reduced protein density and calcification rates. For A. formosa, genetic differences, or long-term entrainment to a highly variable environment, appeared to promote coral survival at the expense of calcification. The response decouples coral survival from carbonate coral reef resilience, a response that was further exacerbated by reductions in irradiance. As we begin to discuss interventions necessitated by the CO2 that has already been released into the atmosphere, we need to prioritise our focus on the properties that maintain valuable carbonate ecosystems. Rapid and dense calcification by corals such as branching Acropora is essential to the ability of carbonate coral reefs to rebound following disturbance events and maintain 3D structure but may be the first property that is sacrificed to enable coral genet survival under stress.Coral reefs are facing increasingly devasting impacts from ocean warming and acidification due to anthropogenic climate change. In addition to reducing greenhouse gas emissions, potential solutions have focused either on reducing light stress during heating, or on the potential for identifying or engineering “super corals”. A large subset of these studies, however, have tended to focus primarily on the bleaching response of corals, and assume erroneously that corals that bleach earlier in a thermal event die first. Here, we explore how survival, observable bleaching, coral skeletal growth (as branch extension and densification), and coral tissue growth (protein and lipid concentrations) varies for conspecifics collected from distinctive reef zones at Heron Island on the Southern Great Barrier Reef. A reciprocal transplantation experiment was undertaken using the dominant reef building coral ( Acropora formosa . Acropora formosa is abundant in diverse zones of a coral reef where it contributes to the 3-dimensional structure and carbonate accumulation within coral reef ecosystems [A. formosa is one of the most dominant branching corals found on Heron Island Reef in the Southern Great Barrier Reef (GBR) of Australia and is present on the reef slope and in lagoons typically [2 mitigation pathways, such as that agreed to in Paris at the United Nations Climate Change Conference in 2015. Increases in atmospheric pCO2, principally due to the burning of fossil fuels, have increased the global temperature by 1°C, and equilibration of atmospheric pCO2 with the oceans has increased ocean acidification by 0.1 pH units, since pre-industrial times [Primary  and secondary  calcification by reef-building corals ,2 is criosystems . A. formypically . A reef ypically  and dailypically ,17. Undesa Dana, 46. Acropypically . Initialypically . Now, moypically ,22. Highypically . A negatal times . In addial times . Ultimat2 emission scenarios such as RCP2.6 [The degree to which these ecosystems will be impacted under reduced COs RCP2.6  is nonets RCP2.6  or via as RCP2.6 ,29. Precs RCP2.6 ,31. Somes RCP2.6 , others s RCP2.6 ,32,33. Is RCP2.6 . In thiss RCP2.6 –36.In literature regarding “super corals”, bleaching susceptibility  is often erroneously assumed to be interchangeable with mortality –39. Furt2), temperature and light intensity, which is associated with the large tidal range (3m) and the resulting ponding of reefs at low tide [Other managerial or adaptive solutions to protecting reefs of the future involve the application of shade to reefs exposed to thermal stress . Given tlow tide ,50,51. Plow tide . Over sulow tide ,54. Survlow tide –57, a prlow tide .2 conditions are thought to be relatively stable over daily time scales due its proximity and exchange with external ocean waters [By contrast, on the reef slope of Heron Island, temperature and pCOn waters . Light, n waters ,62. The n waters . Therefon waters .A. formosa collected from in two distinct habitats. We aimed to test an alternative hypothesis that corals survive highly variable environments because they minimise extension rates that enable them to invest their energy into tissue maintenance and production (change in protein density for fixed extension rates). We further aimed to explore whether there was any influence of prior environmental history  that might constrain the responses of colonies of A. formosa. Specifically, we sought to: (i) determine whether exposure to higher abiotic variability in prior life history increases coral survival irrespective of the characteristic of the new environment; (ii) Whether properties that potentially increase survival are traded for reduced rates of calcification (extension or densification rates); (iii) whether light reductions associated with alteration in depth and reef location, led to a rapid acclimation response  or not and whether a potential decrease in energy acquisition occurs as corals face changed environmental conditions.We ran a reciprocal field experiment on -2s-1, SD = 265.24, Reef flat mean = 394.74 μmol quanta m-2s-1, SD = 564.92). Mean temperature at the reef flat and slope locations remained similar between sites seasonally but was significantly more variable on the reef flat . Heron Reef is dominated by Acropora spp. [Heron Reef 23°27’S, 151°55’E) is a lagoonal platform reef which is part of the Capricorn Bunker group located on the southern end of the Great Barrier Reef, Australia. Past sea surface temperatures (SST) for the sea surrounding Heron Reef had a maximum monthly mean (MMM) of 27.3°C . This i1°55’E isora spp. ,51 whichora spp. .A. formosa were collected from different colonies, roughly 5m apart, at each location  in late September 2017. Fragments were randomly collected from the growing tips of colonies fragments with axial branches or damaged apical corallites were excluded. While these were selected from different colonies to avoid pseudo-replication the colony from which a fragment was collected was not tracked. Corals were kept in flow through aquaria under a shade cloth for 2 weeks during initial measurements and preparation for the different experimental treatments. All fragments were trimmed to 7cm using bone cutter scissors. A total of 25 fragments from each location  were sampled to assess initial coral condition. These fragments were stored at -20°C and analysed at Heron Island Research Station and/or the University of Queensland. A hole was drilled ~1cm from the base of each fragment intended for reciprocal transplantation, in order to attach the coral to pieces of flat live rock (~25cm x 25cm) for outplanting, 20 corals were attached to each rock. Live rock was used to stabilise fragments rather than plastic or metal structures to limit algal growth and potential competition (Fragments (7-10cm) of petition . Fragmenpetition , corals DWwax = dry weight of skeleton with single coat of wax, BWwax = buoyant weight of skeleton with single coat of wax, ∂m = density of the fluid medium.Calcification rates were calculated from initial and final buoyant weight measurements of each individual fragment. Buoyant weight (BW) was measured using methods described by Spencer Davies , and JokThe coral tissue in PBS was centrifuged at 4500rpm for 5 minutes , to separate the symbiont pellet. The resulting supernatant was stored in two parts to allow for protein and lipid analyses. For protein analyses the supernatant was analysed with a spectrophotometer , using absorbance values at 235nm and 280nm. Protein densities were then calculated using equations in Whitaker and Granum . For lipFragments in the reciprocal transplant could only be identified as dead in collection periods , at which point they were removed from the live rock. At the midway point of the study, 24 corals were classified as dead (defined as 100% tissue loss) and removed, with one additional coral removed due to mortality at the end point of the study.Minor tissue dieback (less than 5%) on all live fragments was also assessed at the end of the experiment. Approximately 80% of shallow transplants, and 77% of deep transplants experienced a small  and 75th percentile (Q3) were determined for temperature, 12h (6h – 18h) daytime mean, Q1 and Q3 were determined for PAR.Field environmental variables were measured using temperature , and photosynthetically active radiation  loggers. Three PAR and three temperature loggers were placed at each location  each within 5 metres of the metal frames on which the corals were attached. On the reef slope, two of the three PAR loggers failed and as a result only one set of data was able to be used for analyses. PAR loggers were fitted with a copper washer around the sensor to prevent biofouling and were cleaned at the midway point when corals were collected for measurements. Temperature loggers collected data as hourly means; PAR loggers collected data as integrated values every 2 hours. For analyses, 24h daily means, 25Host protein was used as a proxy for host biomass, as has been used across multiple peer reviewed studies –85. We uAll statistical analyses were conducted with R version 3.6.2 software (2018), figures were created using the ggplot2 package  and sjPlFor physiological analysis, model response variables were: BV, LSA, BW, total protein, and total lipids all of which were continuous. Of the fixed predictors, Origin and Transplant were categorical, while the remaining  were continuous. Due to collinearity as measured through variance inflation factor (VIF), covariates BV and LSA were identified as interchangeable in subsequent models. The model with the best fit was chosen as previously described. All physiological analysis and mortality models included live rock as a random effect, which refers to the live rock on which the coral was placed, this remained constant throughout the experimental period and is representative of the coral’s location in space.The probability of mortality was analysed as a binomial response variable using a mixed effect logistic regression [GLMER, lme4, A generalized least squares (GLS) model was used to compare environmental variables (Temperature and PAR) between each site  over the four-month experimental period. A GLS model was used as data were identified as being temporally autocorrelated. The daily mean, and interquartile range , as an indication of variability, were both analysed for temperature and PAR, full and best fit models are shown in Response and predictor variables for the initial and best fit models applied to the analysis of samples from the reciprocal transplantation experiment conducted on Heron Island (Southern GBR). Underlined predictors in best fit model indicate a significant effect (P<0.05), the direction of these significant effects is indicated in brackets of the best fit models. Continuous predictors such as “BV” (+) defines a positive correlation to the response variable. For categorical predictors such as “transplant” the relationship between levels of the factor are provided; Transplant, Sh = Shallows, D = Deep; Origin, F = Flat, and Sl = Slope. The direction of interaction effects is not indicated in this table. Variables are abbreviated in model outputs; bulk volume = BV, living surface area = LSA, buoyant weight = BW. = 830.900, P = <0.001,  = 3.500, P = 0.062,  = 666.535, P = <0.001,  = 5.680, P = 0.017, Mean temperature increased over time (with the onset of summer) at both sites  = 5.9214, P = 0.0156). Mean daily (06:00–18:00) PAR over the experimental period was always higher in the shallows  than the deep . However, amplitude changes in mean daily PAR over successive days were often greater in the shallows than in the deep  = 5.807, P = 0.0166, A significant interactive effect was observed between date and location for mean PAR  = 15.420, P = <0.001, χ2 = 4.604, d.f. = 1, P = 0.032, Over the length of the experimental period, corals that originated from the reef slope, irrespective of their transplant location, were 20% less likely to survive than reef flat origin corals ; (ii) rates of change in LSA were significantly greater in corals transplanted to reef flat compared to those transplanted to reef slope  found that when other parameters were held constant at mean value: (i) rates of change in LSA correlated positively with rates of change in coral volume  rates of change in BW correlated positively with rates of change in fragment LSA  corals that originated from the reef flat and were transplanted to the shallow  had significantly higher rates of change than any other group  between χ2 = 6.342, d.f. = 1, P = 0.012); (ii) rates of change in total proteins were significantly greater in fragments originating from the reef flat transplanted to the shallow, than transplanted to the deep  rates of change in total proteins correlated positively with rates of change in LSA . But showed no significant effect of fragment origin or transplant location.Relative rates of change in total lipids of Porites cylindrica at the same locations which maintained native properties over 21 months [We hypothesized that long-term exposure to the highly variable reef-flat environment would both increase survival potential and engender a conservative growth response, where greater biomass is accumulated at the cost of conserving primary calcification rates. A conservative response that would be maintained upon transplantation to the new reef slope environment. The results of our study supported our hypotheses, and thereby, suggest that any potential epigenetic memory implanted by exposure to long-term prior stress is unlikely to be a panacea that will save carbonate coral reefs in the short-term. It is highly likely that reef-flat coral are better survivors irrespective of habitat because they have reduced rates of primary extension. In the present study, reef-flat native coral properties were maintained over 5 months but removal to the less stressful reef-slope environment did not coincide with an increase in rates of calcification. These findings are consistent with previous studies using 1 months . Other s1 months  however,1 months . Consequ1 months . The res1 months ,95. In a1 months .The environmental contrast between the reef slope and reef flat is exhibited by the difference between the mean and variability of the recorded PAR and temperature. The high variability in temperature on the reef flat observed in this study is comparable to temperature fluctuations observed in studies identifying specific coral populations with superior thermal tolerance ,96. The Whilst mean temperature between the reef flat and reef slope did not differ significantly, mean PAR did. Based on these data, we conclude that the primary difference between the deeper slope and shallower flat zones is light. However, there could be additional confounding factors such as differences in the concentration of available dissolved or particulate organic matter that were not measured but have been described in other studies –100. TheOver the experimental period of 5 months, reef flat origin corals showed a reduced ability relative to corals originating from the reef slope to acclimate to their new environment. Reef flat origin corals appeared unable to increase their energy acquisition to maintain the additional energy required to increase skeletal and protein densities on the slope, despite their general tendency to exhibit slower volume and tissue expansion rates ,86. ThisA potential reason the corals benefitted from the higher light environment in our study is that the high light habitat is potentially relatively richer in dissolved and particulate organic food ,100. CorA. formosa that showed greater potential to survive a diversity of environments appear to exhibit a reduced ability to acclimate to changes in light availability. Attempting to further protect such corals from bleaching by periodically reducing light fields [We have demonstrated here that conspecific of t fields , would tt fields ,109. Rapt fields ,114, potA. formosa from this study, clipped to the same length, have different volumes and skeletal densities that forced the use of relative changes to preserve the statistical assumption that covariates are independent of treatment effect.On a more positive note, reef flat origin coral transferred back to the reef flat had higher skeletal density relative to other treatments, whilst also maximising protein density. Consequently, primary calcification was reduced. Increases in skeletal density can reduce fragmentation and over the long-term ,116, eveA. formosa populations are driven by epigenetic consequences associated with prior long-term exposure to different abiotic environments within the reef-scape. There is, however, the potential that the relative inflexibility of the two coral populations to alter properties when exposed to new environments has a genetic cause in the form of local adaptation. The two populations may hide cryptic speciation, a phenomenon that is increasingly observed amongst coral taxa [Seriatopora hystrix genetic isolation has been observed to result from differences in reproductive timing between populations in deep and shallow habitats [Pocillopora damicornis [P. damicornis was observed on Heron Reef over similar geographic distances as in the current study. However, P. damicornis is a brooding species while the target species in the current study, A. formosa, is a broadcasting species.Presently, we assume that the differences observed between these relatively proximal ral taxa ,121,122.ral taxa . In thisral taxa , and asyral taxa . In a brhabitats ,127. Mormicornis . InteresCorals that have a prior life history of chronic exposure to highly variable environments tend to be less susceptible to mortality. In this study, we observed that corals fragments raised on the reef flat have a greater survival potential in any environment than conspecifics raised on the reef slope. Daily third quartile temperature (3Q-DHW) accumulated to an 8°C week over the summer on the reef flat. In combination with an increased irradiance for reef slope origin corals moved to the reef flat, the new environment however did not increase bleaching susceptibility of these reef slope origin corals. Rather, they demonstrated that coral expansion in these slope origin corals was suppressed by the lower light conditions under which they appear to nonetheless thrive on the reef slope. The greater survival potential of reef flat origin corals was correlated to their having greater tissue thickness (as measured by protein density) likely facilitated by reduced rates of primary and secondary calcification. Furthermore, these reef flat origin corals failed to acclimate to the lower light levels offered on the reef slope as suggested by a lack of energy to maintain high tissue protein densities despite reduced branch expansion. The fact that reef flat origin corals do not physiologically morph into reef slope origin corals upon relocation to the reef slope and vice-versa suggests that these corals have become epigenetically or genetically distinct populations. This study supports the hypothesis that exposure to a highly variable environment results in “tough” corals, but it identifies that one of the costs of increased survival potential is reduced calcification. These corals therefore lack the properties that are required to maintain healthy and functioning carbonate reefs exposed to increases in the intensity and frequency of disturbance events as a result of climate change. Further, they lack the ability to respond positively to the reductions in light field likely associated with future sea-level rise. Branching Acroporids are important to the 3-dimensional structure and many services provided by carbonate reefs . The pre