ADARs regulate cuticle collagen expression and promote survival to pathogen infection.

BACKGROUND: In all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA-binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking. RESULTS: Here, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure. CONCLUSIONS: Our data uncover a critical role of the C. elegans ADAR family of RNA-binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity.

RESIC: A Tool for Comprehensive Adenosine to Inosine RNA Editing Site Identification and Classification.

Adenosine to inosine (A-to-I) RNA editing, the most prevalent type of RNA editing in metazoans, is carried out by adenosine deaminases (ADARs) in double-stranded RNA regions. Several computational approaches have been recently developed to identify A-to-I RNA editing sites from sequencing data, each addressing a particular issue. Here, we present RNA Editing Sites Identification and Classification (RESIC), an efficient pipeline that combines several approaches for the detection and classification of RNA editing sites. The pipeline can be used for all organisms and can use any number of RNA-sequencing datasets as input. RESIC provides (1) the detection of editing sites in both repetitive and non-repetitive genomic regions; (2) the identification of hyper-edited regions; and (3) optional exclusion of polymorphism sites to increase reliability, based on DNA, and ADAR-mutant RNA sequencing datasets, or SNP databases. We demonstrate the utility of RESIC by applying it to human, successfully overlapping and extending the list of known putative editing sites. We further tested changes in the patterns of A-to-I RNA editing, and RNA abundance of ADAR enzymes, following SARS-CoV-2 infection in human cell lines. Our results suggest that upon SARS-CoV-2 infection, compared to mock, the number of hyper editing sites is increased, and in agreement, the activity of ADAR1, which catalyzes hyper-editing, is enhanced. These results imply the involvement of A-to-I RNA editing in conceiving the unpredicted phenotype of COVID-19 disease. RESIC code is open-source and is easily extendable.

Endogenous siRNAs promote proteostasis and longevity in germline-less Caenorhabditis elegans.

How lifespan and the rate of aging are set is a key problem in biology. Small RNAs are conserved molecules that impact diverse biological processes through the control of gene expression. However, in contrast to miRNAs, the role of endo-siRNAs in aging remains unexplored. Here, by combining deep sequencing and genomic and genetic approaches in Caenorhabditis elegans, we reveal an unprecedented role for endo-siRNA molecules in the maintenance of proteostasis and lifespan extension in germline-less animals. Furthermore, we identify an endo-siRNA-regulated tyrosine phosphatase, which limits the longevity of germline-less animals by restricting the activity of the heat shock transcription factor HSF-1. Altogether, our findings point to endo-siRNAs as a link between germline removal and the HSF-1 proteostasis and longevity-promoting somatic pathway. This establishes a role for endo siRNAs in the aging process and identifies downstream genes and physiological processes that are regulated by the endo siRNAs to affect longevity.

Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing.

A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes.

QsRNA-seq: A protocol for generating libraries for high-throughput sequencing of small RNAs.

Small RNAs (sRNAs) are 20-30 nt long non-coding RNA molecules that regulate essentially all cellular processes. Besides being an intensively studied topic in academic research, sRNAs also hold a promise as clinical biomarkers. While the need for expressional profiling of sRNAs is growing, preparation of sRNA libraries for high-throughput sequencing (HTS) remains technically challenging, due to their small size. The common PAGE-based protocol is time-consuming and inefficient due to material loss, while gel-free protocols generate libraries of insufficient quality. To overcome these shortcomings, we modified the conditions of size-selection by Solid Phase Reversible Immobilization (SPRI) in a way that allows separation of nucleic acids shorter than 100 nt and differing in length by only 20 nt. Implementing the method for preparation of small RNA libraries for HTS resulted in QsRNA-seq, a gel-free, fast and easy-to-perform protocol, amenable to automation, generating very clean libraries that result in high-depth expression data. The protocol also utilizes Unique Molecular Identifiers (UMI) for reduction of library preparation biases and to quantify expression levels. QsRNA-seq provides an excellent solution to the growing needs for small RNA expression profiling for research clinical use.

A-to-I RNA Editing Affects lncRNAs Expression after Heat Shock.

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on double-stranded RNA (dsRNAs). Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant Caenorhabditis elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that long non-coding RNAs (lncRNAs) and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

Escherichia coli mediated resistance of Entamoeba histolytica to oxidative stress is triggered by oxaloacetate.

Amebiasis, a global intestinal parasitic disease, is due to Entamoeba histolytica. This parasite, which feeds on bacteria in the large intestine of its human host, can trigger a strong inflammatory response upon invasion of the colonic mucosa. Whereas information about the mechanisms which are used by the parasite to cope with oxidative and nitrosative stresses during infection is available, knowledge about the contribution of bacteria to these mechanisms is lacking. In a recent study, we demonstrated that enteropathogenic Escherichia coli O55 protects E. histolytica against oxidative stress. Resin-assisted capture (RAC) of oxidized (OX) proteins coupled to mass spectrometry (OX-RAC) was used to investigate the oxidation status of cysteine residues in proteins present in E. histolytica trophozoites incubated with live or heat-killed E. coli O55 and then exposed to H2O2-mediated oxidative stress. We found that the redox proteome of E. histolytica exposed to heat-killed E. coli O55 is enriched with proteins involved in redox homeostasis, lipid metabolism, small molecule metabolism, carbohydrate derivative metabolism, and organonitrogen compound biosynthesis. In contrast, we found that proteins associated with redox homeostasis were the only OX-proteins that were enriched in E. histolytica trophozoites which were incubated with live E. coli O55. These data indicate that E. coli has a profound impact on the redox proteome of E. histolytica. Unexpectedly, some E. coli proteins were also co-identified with E. histolytica proteins by OX-RAC. We demonstrated that one of these proteins, E. coli malate dehydrogenase (EcMDH) and its product, oxaloacetate, are key elements of E. coli-mediated resistance of E. histolytica to oxidative stress and that oxaloacetate helps the parasite survive in the large intestine. We also provide evidence that the protective effect of oxaloacetate against oxidative stress extends to Caenorhabditis elegans.

QsRNA-seq: a method for high-throughput profiling and quantifying small RNAs.

The ability to profile and quantify small non-coding RNAs (sRNAs), specifically microRNAs (miRNAs), using high-throughput sequencing is challenging because of their small size. We developed QsRNA-seq, a method for preparation of sRNA libraries for high-throughput sequencing that overcomes this difficulty by enabling a gel-free separation of fragments shorter than 100 nt that differ only by 20 nt in length. The method allows the use of unique molecular identifiers for quantification and is more amenable to automation than gel-based methods. We show that QsRNA-seq gives very accurate, comprehensive, and reproducible results by looking at miRNAs in Caenorhabditis elegans embryos and larvae.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

In cancer, A-to-I RNA editing can be the driver, the passenger, or the mechanic.

In recent years, A-to-I RNA modifications performed by the Adenosine Deaminase Acting on RNA (ADAR) protein family were found to be expressed at altered levels in multiple human malignancies. A-to-I RNA editing changes adenosine to inosine on double stranded RNA, thereby changing transcript sequence and structure. Although A-to-I RNA editing have the potential to change essential mRNA transcripts, affecting their corresponding protein structures, most of the human editing sites identified to date reside in non-coding repetitive transcripts such as Alu elements. Therefore, the impact of the hypo- or hyper-editing found in specific cancers remains unknown. Moreover, it is yet unclear whether or not changes in RNA editing and ADAR expression levels facilitate or even drive cancer progression or are just a byproduct of other affected pathways. In both cases, however, the levels of RNA editing and ADAR enzymes can possibly be used as specific biomarkers, as their levels change differently in specific malignancies. More significantly, recent studies suggest that ADAR enzymes can be used to reverse the oncogenic process, suggesting a potential for gene therapies. This review focuses on new findings that suggest that RNA editing by ADARs can affect cancer progression and even formation. We also discuss new possibilities of using ADAR enzymes and RNA editing as cancer biomarkers, indicators of chemotherapeutic drug sensitivity, and even to be themselves potential therapeutic tools.

A-to-I RNA editing - thinking beyond the single nucleotide.

Adenosine-to-inosine RNA editing is a conserved process, which is performed by ADAR enzymes. By changing nucleotides in coding regions of genes and altering codons, ADARs expand the cell's protein repertoire. This function of the ADAR enzymes is essential for human brain development. However, most of the known editing sites are in non-coding repetitive regions in the transcriptome and the purpose of editing in these regions is unclear. Recent studies, which have shown that editing levels of transcripts vary between tissues and developmental stages in many organisms, suggest that the targeted RNA and ADAR editing are both regulated. We discuss the implications of these findings, and the possible role of RNA editing in innate immunity.

A-to-I RNA editing promotes developmental stage-specific gene and lncRNA expression.

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3' UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi.

Function of cancer associated genes revealed by modern univariate and multivariate association tests.

Copy number variation (CNV) plays a role in pathogenesis of many human diseases, especially cancer. Several whole genome CNV association studies have been performed for the purpose of identifying cancer associated CNVs. Here we undertook a novel approach to whole genome CNV analysis, with the goal being identification of associations between CNV of different genes (CNV-CNV) across 60 human cancer cell lines. We hypothesize that these associations point to the roles of the associated genes in cancer, and can be indicators of their position in gene networks of cancer-driving processes. Recent studies show that gene associations are often non-linear and non-monotone. In order to obtain a more complete picture of all CNV associations, we performed omnibus univariate analysis by utilizing dCov, MIC, and HHG association tests, which are capable of detecting any type of association, including non-monotone relationships. For comparison we used Spearman and Pearson association tests, which detect only linear or monotone relationships. Application of dCov, MIC and HHG tests resulted in identification of twice as many associations compared to those found by Spearman and Pearson alone. Interestingly, most of the new associations were detected by the HHG test. Next, we utilized dCov's and HHG's ability to perform multivariate analysis. We tested for association between genes of unknown function and known cancer-related pathways. Our results indicate that multivariate analysis is much more effective than univariate analysis for the purpose of ascribing biological roles to genes of unknown function. We conclude that a combination of multivariate and univariate omnibus association tests can reveal significant information about gene networks of disease-driving processes. These methods can be applied to any large gene or pathway dataset, allowing more comprehensive analysis of biological processes.

Co-option of the piRNA pathway for germline-specific alternative splicing of C. elegans TOR.

Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3' splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3' splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a "nonself" intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations.

BAF-1 mobility is regulated by environmental stresses.

Barrier to autointegration factor (BAF) is an essential component of the nuclear lamina that binds lamins, LEM-domain proteins, histones, and DNA. Under normal conditions, BAF protein is highly mobile when assayed by fluorescence recovery after photobleaching and fluorescence loss in photobleaching. We report that Caenorhabditis elegans BAF-1 mobility is regulated by caloric restriction, food deprivation, and heat shock. This was not a general response of chromatin-associated proteins, as food deprivation did not affect the mobility of heterochromatin protein HPL-1 or HPL-2. Heat shock also increased the level of BAF-1 Ser-4 phosphorylation. By using missense mutations that affect BAF-1 binding to different partners we find that, overall, the ability of BAF-1 mutants to be immobilized by heat shock in intestinal cells correlated with normal or increased affinity for emerin in vitro. These results show BAF-1 localization and mobility at the nuclear lamina are regulated by stress and unexpectedly reveal BAF-1 immobilization as a specific response to caloric restriction in C. elegans intestinal cells.

Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis.

Emerin and LEM2 are ubiquitous inner nuclear membrane proteins conserved from humans to Caenorhabditis elegans. Loss of human emerin causes Emery-Dreifuss muscular dystrophy (EDMD). To test the roles of emerin and LEM2 in somatic cells, we used null alleles of both genes to generate C. elegans animals that were either hypomorphic (LEM-2-null and heterozygous for Ce-emerin) or null for both proteins. Single-null and hypomorphic animals were viable and fertile. Double-null animals used the maternal pool of Ce-emerin to develop to the larval L2 stage, then arrested. Nondividing somatic cell nuclei appeared normal, whereas dividing cells had abnormal nuclear envelope and chromatin organization and severe defects in postembryonic cell divisions, including the mesodermal lineage. Life span was unaffected by loss of Ce-emerin alone but was significantly reduced in LEM-2-null animals, and double-null animals had an even shorter life span. In addition to striated muscle defects, double-null animals and LEM-2-null animals showed unexpected defects in smooth muscle activity. These findings implicate human LEM2 mutations as a potential cause of EDMD and further suggest human LEM2 mutations might cause distinct disorders of greater severity, since C. elegans lacking only LEM-2 had significantly reduced life span and smooth muscle activity.

Competition between ADAR and RNAi pathways for an extensive class of RNA targets.

Adenosine deaminases that act on RNAs (ADARs) interact with double-stranded RNAs, deaminating adenosines to inosines. Previous studies of Caenorhabditis elegans indicated an antagonistic interaction between ADAR and RNAi machineries, with ADAR defects suppressed upon additional knockout of RNAi. This suggests a pool of common RNA substrates capable of engaging both pathways. To define and characterize such substrates, we examined small RNA and mRNA populations of ADAR mutants and identified a distinct set of loci from which RNAi-dependent short RNAs are markedly upregulated. At these same loci, we observed populations of multiply edited transcripts, supporting a specific role for ADARs in preventing access to the RNAi pathway for an extensive population of dsRNAs. Characterization of these loci revealed a substantial overlap with noncoding and intergenic regions, suggesting that the landscape of ADAR targets may extend beyond previously annotated classes of transcripts.

Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome.

We have used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of a well-studied genetic model, Caenorhabditis elegans. The three methods include a standard (non-directional) library preparation protocol relying on cDNA priming and foldback that has been used in several previous studies for transcriptome characterization in this species, and two directional protocols, one involving direct capture of single-stranded RNA fragments and one involving circular-template PCR (CircLigase). We find that each RNA-seq approach shows specific limitations and biases, with the application of multiple methods providing a more complete map than was obtained from any single method. Of particular note in the analysis were substantial advantages of CircLigase-based and ssRNA-based capture for defining sequences and structures of the precise 5' ends (which were lost using the double-strand cDNA capture method). Of the three methods, ssRNA capture was most effective in defining sequences to the poly(A) junction. Using data sets from a spectrum of C. elegans strains and stages and the UCSC Genome Browser, we provide a series of tools, which facilitate rapid visualization and assignment of gene structures.

Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma.

Endogenous RNA-directed RNA polymerases (RdRPs) are cellular components capable of synthesizing new complementary RNAs from existing RNA templates. We present evidence for successive engagement of two different RdRPs in an endogenous siRNA-based mechanism targeting specific mRNAs in C. elegans soma. In the initiation stage of this process, a group of mRNA species are chosen as targets for downregulation, leading to accumulation of rare 26 nt 5'-phosphorylated antisense RNAs that depend on the RdRP homolog RRF-3, the Argonaute ERGO-1, DICER, and a series of associated ("ERI") factors. This primary process leads to production of a much more abundant class of 22 nt antisense RNAs, dependent on a secondary RdRP (RRF-1) and associating with at least one distinct Argonaute (NRDE-3). The requirement for two RdRP/Argonaute combinations and initiation by a rare class of uniquely structured siRNAs in this pathway illustrate the caution and flexibility used as biological systems exploit the physiological copying of RNA.