RNA-editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines in structured RNAs. Inosines are read as guanosines by most cellular machineries. A to I editing has two major functions: first, marking endogenous RNAs as “self”, therefore helping the innate immune system to distinguish repeat- and endogenous retrovirus-derived RNAs from invading pathogenic RNAs; and second, recoding the information of the coding RNAs, leading to the translation of proteins that differ from their genomically encoded versions. It is obvious that these two important biological functions of ADARs will differ during development, in different tissues, upon altered physiological conditions or after exposure to pathogens. Indeed, different levels of ADAR-mediated editing have been observed in different tissues, as a response to altered physiology or upon pathogen exposure. In this review, we describe the dynamics of A to I editing and summarize the known and likely mechanisms that will lead to global but also substrate-specific regulation of A to I editing. Adenosine deaminases acting on RNA (ADARs) convert adenosines to inosines in structured and double-stranded RNAs . ADAR acEvolutionary, ADARs are related to Adenosine Deaminases Acting on tRNAs (ADATs), which are tRNA deaminating enzymes that are responsible for the introduction of inosines at the wobble position of certain tRNAs . ADARs aIn mammals, three ADAR family members can be found. The most obvious differences between these three family members are seen in the number of double-stranded RNA-binding domains and in the amino-terminal extensions of the ADAR enzymes. While ADAR1 harbors three dsRBDs, ADAR2 and ADAR3 contain only two dsRBDs. In the amino-terminal regions of the ADARs, additional nucleic acid-binding domains but also regions controlling intracellular localization of the harboring enzymes can be found .In mammals, catalytic activity for ADAR1 and ADAR2 can be detected, while ADAR3 is seemingly inactive . ConsequLoss of ADAR1 or ADAR2, in contrast, leads to dramatic phenotypes. Loss of ADAR1 leads to embryonic lethality in mice that is accompanied by liver disintegration and a dramatic increase in interferon signaling ,7. This The precise function of the constitutively expressed ADAR1 p110 isoform that is predominantly localized to the nucleus is not well understood. Seemingly, ADAR1 p110 cannot compensate for loss of the ADAR1 p150 as the selective deletion of the ADAR1 p150 isoform still leads to embryonic lethality .ADAR2 is most strongly expressed in the nervous system but also in some organs such as the intestine and the vasculature ,18. LossAs indicated above, editing can affect the fate and function of RNAs by several means. On the one hand, the A to I exchange introduced by ADARs can affect RNA folding and the binding landscape of proteins interacting with substrate RNAs . In turnTherefore, like many other modifications of nucleic acids, proteins, lipids or carbohydrates that affect the biochemical activities and cellular functions of these biomolecules, also ADAR-mediated RNA editing needs to be tightly controlled and regulated to assure cellular and organismic homeostasis. Indeed, it was already shown that protein-recoding RNA-editing events vary largely between tissues and throughout development ,25. For Drosophila or cephalopods, the editing levels have been found to correlate with different temperatures. In Drosophila, this change may result from temperature-induced alterations of RNA structures [In invertebrates, such as ructures . In cephructures . Most inructures .In this review, we will focus on the different mechanisms that can affect ADAR-mediated editing. Drosophila melanogaster that correlate with the temperatures in which these flies live [As mentioned above, the double-stranded structures required for RNA editing can show temperature-dependent variability in the case of poikilotherms. Consequently, minor changes in the underlying sequences can equally affect the stability of these double-stranded structures. It is therefore not too surprising that single nucleotide polymorphisms (SNPs) have been detected in variations of ies live . In factAt a more global level, the abundance and conservation of genomic repeats is a major regulator of RNA editing. As inverted repeats within transcripts are major sites of recruitment for ADARs, the repeat repertoire in transcripts not only controls the editing levels of the repeats but also of the adjacent sites ,35,36.Post-translational modifications (PTMs) are known to dynamically modify protein fate and function in eu- and prokaryotes. ADAR proteins are also subject to PTMs that impact on their enzymatic activity and stability.One of the first post-translational modifications found on ADAR proteins was the SUMOylation  of ADAR1 . SUMO-1 Since ADAR1 plays an important role in the attenuation of IFN induction by editing and thereby “masking” self-dsRNAs from recognition by dsRNA sensors, Li and colleagues  postulatBoth active ADARs have been identified as targets of protein phosphorylation mainly in the context of high-throughput phosphoproteomic studies (PhosphoSite). However, to date only few sites have been experimentally proven to affect enzymatic activity of the editases. More recently, the editing activity of ADAR1p110 and ADAR2 was found to be regulated by phosphorylation of specific sites . One sitADAR localization and stability can also be used to regulate editing efficiency in a cell. Some factors that influence either localization or protein stability have been identified to date. ADAR2 is regulated by binding to the prolyl-isomerase PIN1. Binding requires a phosphorylated recognition motif (P-Ser/P-Thr-Pro) in the N-terminal region of ADAR2. Upon binding, PIN1 catalyzes the cis/trans isomerization of the peptide bond, resulting in a conformational change that positively affects the ADAR2 editing activity. In the absence of PIN1, ADAR2 is mis-localized to the cytoplasm and marked for degradation by ubiquitination. The responsible enzyme, the E3 ubiquitin ligase WWP2, binds to two conserved PPxY motifs within the N- and C-terminus of ADAR2 and consequently poly-ubiquitinates ADAR2. This, in turn, leads to the degradation of ADAR2 by the proteasome, and thus a reduction in the ADAR2 protein levels. . The ADARegulation of editing is often required in dynamic processes such as development, tumorigenesis or temperature adaptation. One example for this is the regulation of the nuclear ADAR2 protein levels by importin-α4 during the maturation of neurons. As they mature, more importin-α4 is expressed and interacts with ADAR2, thereby fostering its nuclear localization. Within the nucleus, ADAR2 interacts gradually more with the isomerase PIN1. This way, despite the fact that the protein levels of the ADARs are constant during brain development, the ADAR2 editing efficiency is higher in mature neurons compared to immature ones .Notophthalmus viridescens. These amphibians are known for their great regenerative capacity. Upon injury, the ADAR1 protein is shuffled from the cytoplasm into the nucleus and therefore able to edit nuclear targets, while after complete regeneration ADAR1 is found in the cytoplasm again. This nucleocytoplasmic shuttling of ADAR1 ensures editing of the target RNAs during the repair process [Similarly, ADAR1 seems to be regulated during regeneration, as demonstrated by a study in newts,  process .Despite the fact that the editing levels generally rise during development while the ADAR protein levels stay relatively constant, editing regulation is also found at the level of transcription. However, only in certain tissues or cell types. For instance, in hippocampal neurons, the transcription factor CREB (cAMP response element-binding protein) induces expression of ADAR2, thereby protecting them from forebrain ischemia . SimilarHuman ADAR1 harbors a nuclear localization signal that is composed of a bipartite motif that flanks the third double stranded RNA-binding domain . InteresADAR2 harbors an N-terminal nuclear localization signal and is exclusively localized to the nucleus . SimilarThus, the localization of ADARs may modulate enzymatic activity. The NES-bearing cytoplasmic ADAR1-p150 isoform is expressed from an interferon-regulated promoter . InflammEditing occurs predominantly in the nucleus and happens co-transcriptionally. The very same is true for  splicing. All are dynamic processes that not only expand the diversity of the transcriptome but also influence each other. Alternative splicing can regulate editing by producing different ADAR isoforms, which enhance, diminish or abolish editing activity ,60,61,62Especially for the ADAR2 transcript, a number of alternative splicing options have been documented. While not all of them are well conserved, most of them influence either the ADAR protein levels by introducing premature stop codons (PTC) or they affect editing efficiency directly. The inclusion of an alternative exon comprising an Alu (exon 5a) reduces the editing efficiency of ADAR2. Skipping of exon 2, which contains two of the dsRNA binding domains, results in a frameshift that introduces a premature stop. Further, by editing its own mRNA, ADAR2 can autoregulate itself. Self-editing creates an alternative 3´splice site that leads to an insertion close to the translational start site and introduces a premature stop codon ,64.The splicing patterns of ADAR1, apart from its different promoters for interferon-inducible p150 and constitutively expressed p110, are less complex than for ADAR2. One study reported an alternative intron within exon 2 of ADAR1, which is either part of the 5´UTR for ADAR1p110 or within the ORF for ADAR1p150 and leads to a PTC. However, this short transcript seems to be left untouched by nonsense-mediated decay .Since RNA editing and splicing basically appear at the same time and location, it can be expected that these two processes have extensive crosstalk. Thus, it is not surprising that splicing is able to regulate editing. This regulation depends on the efficiency of the splicing process. For editing sites that are defined at least in part by intronic regions, the editing levels are reduced, if the intron is spliced out quickly . On a geVery recently, another more direct mode of regulation of ADAR-editing efficiency was demonstrated, which is brought upon by a change in intracellular pH. The authors could show that intracellular acidification modulates ADAR catalytic activity directly. This is possible due to enhanced flipping out of the adenosine to be edited and additionally an increased deamination rate at a more acidic pH  Figure .trans regulators or competitors of active ADAR proteins comes from within the family. ADAR3, although not an active editase, has a high sequence similarity to ADAR1 and ADAR2 and additionally contains a unique R-domain that allows it to bind ssRNA. ADAR3 is exclusively expressed in the brain, and there it is negatively correlated with overall editing levels [Gria2) mRNA in vivo. In this example, ADAR3 binding to Gria2 mRNA also leads to the inhibition of editing [One of the g levels . In vitrg levels . ADAR3 i editing .Drosophila and human brain [Dm Maleless and its human homolog DHX9, in splicing coordination and cancer, respectively [In a cell with hundreds of different RNA-binding proteins, the heat is on each single RBP to bind to its target RNAs without being outcompeted by other RBPs that can bind as well. This very much depends of course on the spatial and temporal abundance of certain competing proteins. In that sense, most editing regulators identified were specific to only a set of target sites. These site-specific regulators include Fragile X proteins, FMRP and FXR1P, in an brain ,70, and ectively ,72. Other RBPs with clear site-specific regulation of editing are RPS14, SFRS9 and DDX15 regulating ADAR2 targets , and Ro6More recently, two additional interactors of ADAR proteins were identified that regulate editing by RNA binding: The first, a zinc finger RBP, Zn72D, was identified as a regulator of ADAR in the fly brain. Zn72D interacts with ADAR in an RNA-dependent manner and enhances editing of roughly two thirds of the tested editing sites. Its ADAR regulatory function is conserved as the mouse homolog, Zfr, also interacts with and regulates ADAR2 and, to a lesser extent, ADAR1 in mouse primary neurons ,78. One However, the exact binding sites of these RBPs and thus their mode of action is still missing. Additionally, one has to keep in mind that effects of most of these RBPs on editing greatly depend on the cell or tissue type. This is nicely demonstrated, among others, by the study of Quinones-Valdez and colleagues that looked at RBP-KDs in two different cell lines (K562 and HepG2), resulting in two different and only partially overlapping sets of ADAR1 regulators.The dynamic regulation of RNA editing strongly suggests that A to I deamination can respond to environmental or physiological changes. Such changes in the surroundings of cells and organisms will obviously need to be translated into biochemical signals, which have been outlined above. Still, at this point, the pathways that relay extracellular signals into editing patterns are not understood. To this end, signals that can affect editing include temperature , social Understanding editing regulation is important for therapeutic approaches. Today, ADARs are exploited as tools to repair and alter the coding information of RNAs for therapeutic means. To do so, ADAR-derived constructs can be introduced to cells that are targeted to specific substrates. However, another approach is the recruitment of endogenous ADARs to specific target transcripts by artificially generating double-stranded structures using antisense oligonucleotides. Here, it is still not clear which ADAR isoform is best suited to be recruited to the site of editing. Obviously, the enzyme to be recruited needs to be expressed in the respective tissue or cell type. Since the expression of ADAR2 is relatively restricted, it seems that ADAR1 and its isoforms may be more suitable to direct site-directed editing. Still, the different intracellular localizations and modifications of the proteins may be important factors to be considered as the antisense RNA will need to be delivered to the same cellular component.


These four major topics are represented in 23 review articles which are generated from the plenary presentations being delivered by multiple speakers. The content of these review articles is further discussed in the ensuing commentary on Introduction to this Special Issue: Novel Frontiers in Cancer Metastasis.The 8th International Cancer Metastasis Congress being held between October 25–27, 2019 in San Francisco . We are grateful to the Editorial Board of the Clinical and Experimental Metastasis to allow us to publish the presentations of the 8th International Cancer Metastasis Congress in this Special Issue. We are indebted to all the presenters and authors, who have summarized their presentations as review articles, which are separately published online and have been combined into this Special Issue, Novel Frontiers in Cancer Metastasis [We appreciate our sponsors and exhibitors who have made this Congress a success. They are acknowledged in our website (tastasis .


Modifications in nucleic acids are present in all three domains of life. More than 170 distinct chemical modifications have been reported in cellular RNAs to date. Collectively termed as epitranscriptome, these RNA modifications are often dynamic and involve distinct regulatory proteins that install, remove, and interpret these marks in a site-specific manner. Covalent nucleotide modifications-such as methylations at diverse positions in the bases, polyuridylation, and pseudouridylation and many others impact various events in the lifecycle of an RNA such as folding, localization, processing, stability, ribosome assembly, and translational processes and are thus crucial regulators of the RNA metabolism. In plants, the nuclear/cytoplasmic epitranscriptome plays important roles in a wide range of biological processes, such as organ development, viral infection, and physiological means. Notably, recent transcriptome-wide analyses have also revealed novel dynamic modifications not only in plant nuclear/cytoplasmic RNAs related to photosynthesis but especially in chloroplast mRNAs, suggesting important and hitherto undefined regulatory steps in plastid functions and gene expression. Here we report on the latest findings of known plastid RNA modifications and highlight their relevance for the post-transcriptional regulation of chloroplast gene expression and their role in controlling plant development, stress reactions, and acclimation processes. The chloroplast is the result of an endosymbiotic event in which a cyanobacterium was ingested by a eukaryotic host cell. Although chloroplast biogenesis requires regulation of transcription rates, umpteen posttranscriptional events predominate in the development- and environment-dependent control of gene expression ,2,3,4,5.Recently, methodological advances in next-generation sequencing including nanopore sequencing, CLIP-technologies, antibody-based approaches, mass spectrometry, ribosomal profiling, as well as the use of chemical and enzymatic modifications have significantly increased our knowledge about epitranscriptomics and laid the foundation for understanding its role in regulation of gene expression in prokaryotes and eukaryotes .More than 170 different modifications in coding and non-coding RNAs as well as several hundred factors involved in epitranscriptomics have been discovered ,16,17. T6A, m1A, m7G, m5C or hm5C, adenosine dimethyltransfer (m6Am) [+ cap, the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) of tRNAs, and many others   as well y others . All theOur knowledge about the function of chloroplast epitranscriptomic activities is still in its infancy. Forthcoming state-of-the-art ‘omics’ technologies will considerably improve the monitoring of RNA modification networks at the genomic scale in the plant genetic compartments and will certainly shed light on the function and regulation of epitranscriptomic players. In this review, we discuss our present knowledge about novel methods for the identification of RNA modifications, bioinformatic tools, and the potential physiological roles of RNA modifiers and interpreters in plant nuclear/cytoplasmic gene expression related to photosynthesis and the post-transcriptional fate of chloroplast RNAs.Immunoprecipitation of modified RNAs in combination with high-throughput sequencing, mass spectrometry-based techniques, and nanopore sequencing are the three most common approaches for the transcriptome-wide detection and quantification of epitranscriptomic marks. Some of the methods are further combined with chemical or enzymatic steps to identify signature-based marks in RNA molecules . However1A, m6A, and m5C and other base modifications, several studies of the cellular epitranscriptome in Arabidopsis focused on immunological approaches, such as the predominant N6A-methylated RNA immunoprecipitation sequencing (MeRIP/m6A-seq). This technique requires significant quantities of RNAs, high quality antibodies with little cross reactivity, and involves immunoprecipitation of about ~100 nucleotides-long RNA fragments using m6A-specific antibodies, followed by sequencing of the immunoprecipitated fragments or the corresponding cDNAs [6A can potentially also detect a second base modification, N6,2-O-dimethyladenosine (m6Am), which is located at the 5’ end of transcripts. In addition, the mandatory fragmentation of RNAs for library constructions may result in the underrepresentation of m6A sites [6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) can be used to induce specific mutational signatures that allow for the precise identification of m6A residues in RNA molecules. In this approach, antibodies raised against m6A are UV-crosslinked to RNAs and subsequent reverse transcription of crosslinked RNAs results in a precise pattern of mutations or truncations in the cDNA. These signatures are computationally identified and allow mapping of m6A residues at single-nucleotide resolution [Since antibodies are available for mng cDNAs . However6A sites . To over6A sites ,30,31,32solution .1A, m3C, and m1G, can easily be detected because they induce reverse transcription errors or termination, as compared to their unmodified sites. For the detection of m5C, RNA can also be treated with sodium bisulfite prior reverse transcription. This chemical compound deaminates cytosine to uracil resulting in a thymine during reverse transcription. In contrast to the unmethylated cytosine, the m5C methylation is not prone to this deamination and thus can be detected.Sequencing approaches in epitranscriptomics rely on cDNAs as template. A weak point of this method is that some of the epitranscriptomic information is lost when immunoprecipitated RNA is reverse transcribed. However, some of the modification marks, such as m6A is based on the complete enzymatic digestion of the RNA into individual nucleotides or nucleosides followed by various LC–MS/MS (liquid chromatography coupled to tandem mass spectrometry)-based methods [m/z) to estimate their molecular mass and abundance. The modified RNA digestion products can readily be identified due to an increased molecular mass compared with the unmodified standards. This method plays an important role in the discovery, simultaneous detection, and quantification of many different RNA modifications. However, despite its high sensitivity, accuracy, and low detection limit in the fmol to amol range, co-eluting compounds with the identical masses, such as the modifications m1G and m2G, cannot be distinguished. Importantly, due to the complete digestion all information about the sequence context and co-occurrence of modifications is entirely lost. To overcome this shortcoming, RNA molecules are partially digested using specific RNases. The resulting RNA oligonucleotides are then analyzed by MS/MS and the covalent modifications are recognized with single-nucleotide resolution based on the molecular mass shift. A comparison with mass spectra provided by a sequence database enables the determination of the precise position and nature of modifications within the oligonucleotide that can be determined using a computational platform for high-throughput data mining [The prerequisite for classical mass spectrometry in the detection of diverse RNA modifications including m methods . After ia mining .Nanopore direct RNA sequencing (DRS) can be applied to measure and quantify many but not all RNA methylations at defined positions without fragmentation or amplification. This technology relies on a protein nanopore that resides in a membrane through which an electrical current is created. The RNA sequence can be identified by the magnitude of signals transmitted when intact RNAs pass through the nanopore by a motor protein . The use6A methylation marks in nuclear-derived RNAs are often related to chloroplast function, thus we will provide a brief overview of the nuclear/cytoplasmic m6A methylome with a focus on this organelle (6A), the mainly nuclear localized modifiers (methyltransferases and demethylases) and mostly cytoplasmic interpreters are called writers, erasers, and readers, respectively [6A writer complex consists of several conserved and essential components in eukaryotes, including the methyltransferases MTA and MTB, the splicing factor FKBP12 Interacting Protein37 (FIP37), VIRILIZER (VIR), and the conserved E3 ubiquitin ligase HAKAI [vir-1 mutant defective in nuclear m6A methylation and VIR-complemented lines in Arabidopsis [ptm intron 10 terminates transcription prior to a sequence encoding the transmembrane domain, consequently bypassing established retrograde control [6A modifications in the VIR-complemented line were identified. The AAm6ACU and AAm6ACA motifs were confirmed to be the most frequently detected m6A marks in Arabidopsis [6AUU were also detected, raising the possibility that a C following m6A is not a constant feature of the Arabidopsis m6A code [6A in cytoplasmic mRNAs was also confirmed. The differential error sites were exclusively found in this region and no enrichment over stop codons was identified using both, miCLIP or DRS. Strikingly, the impact of m6A loss on pre-mRNA processing was determined and a clear defect in RNA 3’ end formation in vir-1 was observed [vir-1 mutant were identified. For instance, the prpl34 mRNA, which encodes a chloroplast ribosomal protein, is methylated in at least two positions in the 3’ UTR and displays an increase in alternative polyadenylation at a proximal poly(A) site in the vir-1 mutant. These findings suggest that changes in 3’ end poly(A) position of RNAs in the vir-1 mutant may result directly from the loss of m6A and implies a crucial role of the cellular m6A methylome in plastid gene expression.Recent data revealed that mrganelle . With reectively . The nucbidopsis . Frequenbidopsis ,38. Clea control . 17,491 bidopsis ,40,41. Sm6A code . The preobserved . 3579 ge6A distribution within the plant mRNA molecules, can be solved using state-of-the-art approaches and new technologies. Still, it is important to restate that the effects of m6A are challenging and possibly condition-dependent. To date, many chloroplast-related, nucleus encoded transcripts that carry m6A were identified . Furthermore, about 4.6 to 5.8 m6A sites per transcript were found in the chloroplast but only about 1.4 to 2.0 sites per transcript in the cytoplasm, again emphasizing an important link between m6A modifications and chloroplast functions [Remarkably, chloroplast transcripts are highly mpression ,43. Overunctions . The mos6A enrichment within the 3´ UTR and near stop codons in nuclear-derived mRNAs was not observed in the chloroplast. Instead, m6A peaks were found evenly distributed in chloroplast transcripts with higher methylations in exons when compared to introns, suggesting that the regulatory mechanisms may be different between the nucleus/cytoplasm and chloroplast systems. The translation and stability of chloroplast transcripts are commonly regulated by factors acting on 5’ and 3’ UTRs. In addition, degradation of plastid RNAs is thought to be initiated by endonucleolytic cleavages [6A methylation in conjunction with RNA-binding proteins controls the fate of mRNAs in the plant organelle at multiple levels.The previously observed dominant mleavages ,52. Thus6A consensus sequences in the chloroplasts and mitochondria share homology to those in mammals and plant nuclear transcritptomes, indicating an evolutionary related process [6ACC and GGm6ACU. The methylation extent compared to RNA levels in the chloroplast was similar to that found in the nuclear-derived RNAs in Arabidopsis, as most of the highly expressed plastid transcripts were less modified by m6A, and vice versa, corroborating a related or analogous development. However, tissue-specific deviations were also observed. For example, in root amyloplasts the moderately expressed transcripts were more methylated and those expressed at lower or higher levels carried less m6A modifications [Most surprisingly, many m process ,51. The ications .7G at tRNA(Met) [6A and m1G at position 37 of tRNA(Cys) [6A, m2G and m7G at tRNA(Ile) [7G at tRNA(Leu) [2A at tRNA(Met) [tRNAs are vital components of the translation machinery and more than 100 tRNA modifications are known so far, which all seem to be associated with translation efficiency, accuracy, and preventing ribosomal frameshifting. Modifications are often conserved and occur at specific sites of distinct chloroplast tRNAs, such as mRNA(Met) , i6A andRNA(Cys) , t6A, m2RNA(Ile) , m7G at RNA(Leu)  and m2A RNA(Met) , pseudouRNA(Met) .To read all possible 64 triple codons, a minimum of 32 tRNAs is needed. However, plant plastids contain less than 32 distinct tRNAs, suggesting that tRNAs with U in their wobble position might pair with any of the four bases at the third position of the codon via superwobble . The basgid) operon encodes the two GidA and GidB enzymes essential for the biosynthesis of mnm5s2U of tRNAs and the S-adenosyl-L-methionine (SAM)-dependent methylation of the 16S rRNA in the highly conserved 530 loop important for ribosomal function, respectively [The bacterial glucose-inhibited division  by TrmE  together with GidA [In ith GidA ,66 and tith GidA . GidA anith GidA .OLPDD, containing a 6-bp deletion as well as 28 SNPs in its ORF, was incapable of forming homodimers in introgression lines and showed a reduced GTPase activity. NIL-OLPDD plants showed multiple developmental defects accompanied by decreased levels of proteins involved in photosynthesis and ribosome biogenesis. Investigations of the tRNA modifications by LC-MS/MS revealed that modification levels of mnm5s2U were significantly reduced in NIL-OLPDD as compared to the WT.Near-isogenic lines generated by introgression and mutants in rice exhibited a pleiotropic phenotype with reduced chloroplast protein levels and altered gene expression that highly affects retrograde signaling . Map-basOLPDD. While the function and biosynthesis of the mnm5s2U modification in bacteria seems to be relatively well understood, the roles of mnm5s2U in chloroplasts remains elusive.Moreover, altered chloroplast transcription and translation in PDDOL seems to activate retrograde signaling, as expression levels of the photosynthesis-associated nuclear genes were significantly reduced in It is very interesting that natural variations of a chloroplast tRNA-modifying enzyme led to severe pleiotropic defects that not only function in chloroplast biogenesis but also in plant development and demonstrates a fast-evolving plastid RNA metabolism despite its conserved mechanism . Whether6A) in all kingdoms of life. The i6A modification can be modified further to 2-methyl-thio-N6-isopentenyladenosine (ms2i6A) and is believed to stabilize the Watson-Crick base pairing by base stacking. The isopentenylation is catalyzed by a conserved isopentenyl transferase important for translation accuracy, efficiency, and non-sense suppression [The 37th base of a subset of tRNAs next to the anticodon can be modified by bulky additions including N6-isopentenyl adenosine  is a well-characterized RNA modification which has been found in high amounts in abundant RNAs in both prokaryotes and eukaryotes  within chloroplasts [mnslation ,79,80. Uroplasts ,82. TRM4E. coli there is a unique dimethyl modification, N4, 2′-O-dimethylcytidine (m4Cm) located at position 1402, which was discovered more than half a century ago. m4Cm1402 plays a role in fine-tuning the shape and function of the P-site, thus increasing decoding fidelity. Two distinct SAM-dependent RNA methyltransferases mraW (rsmH) and yraL (rsmI) are responsible for the N4 and 2′-O-methylations of C1402, respectively. The dimethyl modification of C1402 is conserved in bacteria and plastids [E. coli. This methylation is accomplished by the conserved chloroplast ortholog of RsmH, called CMAL [At the P-site of the 16S rRNA of plastids . Using bled CMAL . CMAL isled CMAL . CMAL-meled CMAL . This idE. coli encodes an rRNA adenine dimethyltransferase responsible for the specific dimethylation at the N-6 position of the base moiety of two adjacent adenosines, A1518 and A1519 (m26A1518m26A1519), located near the 3′-end of the 16S rRNA [E. coli 16S rRNA and thus to complement KsgA E. coli mutants, confirming the conserved function [E. coli led to mild reduction of translation rates and growth, while disruption of DIM1 in yeast is lethal [E. coli KsgA, DIM1 is additionally required for the pre-rRNA processing at cleavage sites A1 and A2 of the 18S rRNA [The KsgA gene in 16S rRNA . This rRfunction . Howevers lethal . The rea18S rRNA .pfc1 mutant and RNAi lines showed chlorosis and impaired chloroplast development when exposed to cold (5°C) but were indistinguishable from the WT when grown at 22°C. Sequence analysis revealed that PFC1 indeed encodes a conserved rRNA methylase required for rRNA adenine demethylation , was identified in a screen for chilling-sensitive Arabidopsis mutants . The pfchylation . Primer 6A methylation in plants in general and in chloroplasts in particular, more in-depth studies are needed. The photoautotrophically viable pfc1 mutant seems to be a good candidate for exploring whether m6A dimethylases, such as PFC1, act alone or in a complex together with other factors and whether the methylation is reversible. Last but not least, the question how PFC1 as an rRNA adenine dimethyltransferase (rAD) mediates chilling-resistance appears an exciting topic and needs to be addressed in the future , but the precise molecular function of most RNA ribose methylations is still not understood [Among many other RNA species eukaryotic rRNAs and snRNAs are known to be highly modified by abundant 2′-derstood . It has derstood . By applderstood . The posBacillus subtilis and polynucleotide phosphorylase (PNPase) were shown to be responsible for RNA 3’-tail synthesis in bacteria. Besides the potential to add A, G, C, and U residues to an RNA chain, PNPases are also known for their processive 3’–5’ degradation activity. The RNA chaperone Hfq stimulates initiated polyadenylation, thereby increasing the processivity of PAP. RNA degradation involves a large set of exo- and endonucleases as well as the action of the eubacterial degradosome [In contrast to the mostly stabilizing role of poly(A) tails at the 3’ ends of cytoplasmic RNAs in eukaryotes, polyadenylation in eubacteria, chloroplasts, and mitochondria as well as in nuclei of diverse eukaryotes promotes the rapid exonucleolytic degradation of RNAs . Severalradosome .In cyanobacteria and chloroplasts polyadenylation and exonucleolytic degradation is performed by PNPase and no enzymatically active PAP I homolog has been identified . KnockdoRNA polyadenylation in chloroplasts is presumably not the initial step in regulation of RNA degradation. Endonucleolytic degradation of plastid RNAs is performed by numerous RNases including RNase J, CSP41a/b, and a degradosome-like complex consisting of RNase E, the rho-containing RHON1 protein, helicases, and additional components ,3,6,102.The majority of 3’ tail addition sites coincided with endonucleolytic cleavage sites indicating that RNA degradation and intron removal is initiated by endonucleolytic activities that create substrates for polyadenylation and that stable stem-loop structures may prevent not only degradation but also polyadenylation ,96,99. OEscherichia coli [Although we already know so much about the mechanisms of plastid RNA degradation and the factors involved, the role and regulation by environmental factors and endogenous influences is virtually unknown. Similarly, nothing is known about the time course of the initial regulation of RNA stability potentially driven by environmental and endogenous factors that could modulate endonuclease activity and/or polyadenylation and thus regulate RNA half-life within chloroplasts. In this context it is worth noting that downregulation of the chloroplast PNPase had only minor effects on mRNA levels, implying that other mechanisms than polyadenylation are key players in the regulation of mRNA steady-state levels . An indihia coli .In contrast to housekeeping transcripts, mostly mRNAs in the fucoxanthin dinoflagellate are edited and/or contain 3’-poly(U) tail extensions. Both posttranscriptional modifications were acquired following serial endosymbiotic events. Many of the characteristics were also found in peridin dinoflagellates whose plastomes consist of many mini-circles encoding one or few photosynthetic proteins. However, other dinoflagellates are lacking both modifications, demonstrating that polyuridylation was acquired by a common ancestor in the fucoxanthin dinoflagellate lineage . Data suIn a recent study poly(U) tails were also found in plastid RNAs within the Cladophorales green alga lineage . Thus, pWith the noticeable exception of budding yeast, uridylation at different nuclear and cytoplasmic RNA populations is catalyzed by terminal uridylyl transferases or poly(U) polymerases in eukaryotes where it is associated in a positive or negative way with RNA degradation, maturation of microRNAs, and the maintenance of U6 small nuclear RNAs . With thElucidating the factors, targets, and possible interacting partners involved in plant organelle polyuridylation, its temporal and spatial regulation and its implication in regulation of gene expression under changing conditions will be a major task as virtually nothing is known about it .Pseudouridine  is a naturally generated modified nucleoside that plays a central role in all kingdoms of life. Most studies performed preferentially in mammals, yeast, and bacteria demonstrated an important function of post-transcriptional pseudouridylation in stabilizing RNA secondary structures that affect RNA localization, ribosome assembly as well as efficiency, and fidelity of protein biosynthesis to regulate growth, development, and responses to various stresses .So far, little is known about the function and components involved in the Ψ modifications in plant cytoplasmic and organellar RNAs ,112. Ψ cPseudouridine is the most abundant nucleoside modification across all RNA species amounting up to 0.4% of all uridines and is prevalently found in partially conserved positions in rRNAs, snRNAs, and the TΨC-loop (Ψ55), the D stem and the anticodon stem and loop of tRNAs but generally to a lesser extent in mRNAs ,110,111.Ψ writers are synthases (PUS enzymes) that use aspartate as the nucleophile. Based on their amino acid sequences, Ψ synthases were grouped into the five families RluA, RsuA, TruA, TruB and TruD, which all modify specific, non-overlapping targets . They caAlthough the functions of most Ψs are still enigmatic, it is generally believed that they primarily serve a structural role in the respective RNAs. Besides profound phenotypic effects, Ψ mutants often show no obvious phenotypic deviations from the WT, implying also regulatory roles of Ψ. Interestingly, Ψ was found to undergo dynamic changes in response to heat and to affect RNA levels in yeast . Moreovewww.arabidopsis.org, accessed on 23 July 2021).Despite increasing knowledge, still little is known about the precise mechanisms and functions of RNA pseudouridylation and proteins involved in plants. Out of 21 putative pseudouridine synthases encoded in the Arabidopsis genome, 8 were annotated as chloroplast enzymes, indicating an important function of Ψ in this organelle  fulfills all the sequence criteria of a bona fide Ψ synthase and was identified as a suppressor of the bidopsis . Eleven proteins . Thus, thenotype . Althougarvation . TCD3 inThe chloroplast protein MAA2 in Chlamydomonas is related to Ψ synthases and has been recruited for the assistance of a Ψ-independent group II intron trans-splicing by an unknown mechanism. However, it remains unclear whether the enzyme function has been maintained within the organelle .The translation of genetic information from DNA to protein requires an intermediate product, the RNA, that generally follows the DNA sequence. However, in some cases the sequence of the RNA does not match the sequence of the cognate DNA. The post-transcriptional processing step, in which RNA bases are altered relatively to the corresponding DNA sequence is known as RNA editing . This prRNA editing was first discovered in 1989 in mitochondria ,127,128.ndhB transcript variants. 5’ and 3’ deletions or nucleotide substitutions adjacent to editing sites within ndhB transcripts provided evidence that cis-acting elements located in the -12/-2 region upstream of the editing site are essential for RNA editing [trans-acting factor required for plastid RNA editing, the pentatricopeptide repeat protein (PPR) CRR4 [CG to AUG, creating the correct ndhD initial codon. Since then, numerous other editing mutants and factors have been described revealing that PPR proteins are key players and the specificity factors for organellar RNA editing (characterized plastid PPR editing factors are summarized in First steps towards the understanding of the determinants for plastid RNA editing came from a transplastomic approach in tobacco plants expressing chimeric  editing . About oPR) CRR4 . The CRRPR) CRR4  and faciThe PPR protein family was first described in Arabidopsis and consists of about 450 members . PPRs arE. coli, where E/E-DYW-containing PLS-PPR proteins from Physcomitrella patens were shown to be able to perform efficient and targeted C-to-U editing in vivo [However, what is the mechanism of RNA editing? It is well understood that the RNA to be edited is specifically recognized and bound by the PLS tract of the PLS-class PPR protein singling out the correct target mRNA. The PLS-PPR protein binds the RNA target in a way that its last PPR motif is associated with the fourth nucleotide upstream of the editable C residue. The PLS tract is followed by the E domains that show similarities to the PPR repeats and are thought to either engage in protein-protein interaction or to contribute to RNA base recognition , however in vivo . Shortly in vivo . A modelPhyscomitrella patens organellar editing seems not to require the interplay of factors other than DYW-PPR proteins, mutant analyses in angiosperms suggest that the editing machinery of higher plants is more complex and relies on additional trans-acting proteins. For example, the previously mentioned factor CRR4 is a truncated PLS-PPR protein that lacks the DYW domain and thus belongs to the so-called E-class proteins. To be able to catalyze the C deamination of the ndhD-1 editing site, CRR4 requires the interaction with DYW1, a small DYW domain-containing protein missing a PPR tract [nuwa and dyw2 mutants are targets of E-type PPR proteins [Although PR tract . Two othproteins . It appeThree other protein classes, that in contrast to PPR proteins act rather unspecifically, were identified as essential components of the plant organellar editosomes: The Multiple Organellar RNA editing Factors (MORFs) , proteinIt also remains unknown how PPR proteins form a functional editosome and whether associated non-PPR proteins, such as RNA chaperones, are generally involved in unwinding stable RNA structures to allow access to the targets or to fulfill regulatory functions ,155.Taken together, increasing mutagenic environmental conditions forced the acquisition of PPR proteins with editase activity in order to reverse the DNA mutations in plant organelles. Many other non-PPR factors co-evolved to support organellar editing of flowering plants, but their functions are still under investigation.


Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1. Retroviruses are unique in that they package their single-stranded RNA genomes as non-covalently linked dimers. Genome dimerization is evolutionarily conserved in retroviruses; mutation of the genome dimerization site or failure of a genome to appropriately dimerize results in loss of infectivity of the resulting virus ,3,4,5,6.In vitro experiments have identified two regions of the genome that are important for retroviral genome dimerization: the dimer linkage structure (DLS) and the dimerization initiation site (DIS). The DLS was identified in Rous sarcoma virus (RSV) and characterized by electron microscopy as a physical region where the 5′ untranslated region (5′UTR) of two partially-denatured RSV genomes interacted to form a dimer structure ,15,16,17Previous studies in cell culture have characterized two models of genomic RNA dimerization in retroviruses. In murine leukemia virus (MLV), genome dimerization occurs in the nucleus in a co-transcriptional fashion, resulting in the preferential formation of genetically identical genome homodimers ,27,28,29To address these questions, we used single molecule RNA labeling methods to visualize RSV unspliced vRNAs in cells and virions. We were interested in determining the subcellular localization of unspliced vRNA heterodimers that were genetically distinct and therefore distinguishable using different fluorescence markers. Notably, vRNA homodimers could not be reliably differentiated from single vRNA molecules (monomers) using these methods because they both appeared as a single-colored focus within the cell.Using confocal microscopy, we detected RSV RNA heterodimers in the nucleus, cytoplasm, and at the plasma membrane, suggesting that genome dimerization may originate in the nucleus, similarly to MLV. Deletion of the DIS from the RSV leader sequence significantly reduced colocalization of genetically distinct unspliced vRNAs, indicating that these dual-colored foci were likely formed by the process of dimerization. Single virion analysis of genome composition demonstrated that RSV primarily packaged genomic RNA homodimers, once again similar to that of MLV. Lastly, we demonstrated that intracellular heterodimers were primarily composed of a pair of unspliced vRNAs rather than being influenced by the presence of a mixture of labeled unspliced and spliced vRNAs. Taken together, these findings define the genome dimerization preferences for RSV and suggest that dimers may initially arise in the nucleus, in an analogous fashion as MLV.pol gene and substitution of the pATV8 gag gene. The pRC.V8 plasmid only contains one splice acceptor site and generates one unspliced RNA and one spliced env RNA ,58mCherrem-loops , with a em-loops ,47 to vimCherry sequence does not interfere with virus assembly, as each of the proviral constructs used was budding competent and can generate virus particles detected by immunoblotting of cell lysates and supernatants  unspliced RNA heterodimers and single-color RNA foci (red or green) per cell were measured using each of the three imaging combinations . The totTo examine where genomic RNA dimerization occurs within the cell, the average number of genomic RNA heterodimers present in the nucleus, in the cytoplasm, and at the plasma membrane was quantitated . ColocalmCherry RNA sequence or a cassette of 24 copies of MS2 stem-loops was inserted into the intron to label unspliced vRNA   C. Two imTo determine whether RSV has a packaging preference for genome homo- or heterodimers, single virion imaging analysis was performed ,32. EquiAssuming random formation of genomic RNA dimers based on the Hardy–Weinberg equation, we would have expected to observe 50% homodimers and 50% heterodimers ,27,30,32env mRNA) [mCherry sequence to the env spliced subgenomic mRNA sequence, we selectively imaged only unspliced vRNAs versus both unspliced and spliced vRNAs  . Splicednv mRNA) , so we wed vRNAs A. Confoced vRNAs B.n = 19 cells and 24 cells, respectively; p = 0.2132; On a single cell basis, the average number of heterodimers per cell was 3.79 ± 0.49 for unspliced vRNA and 2.79 ± 1.10 for spliced plus unspliced vRNAs  contributed to genome dimerization, as the sequences have been used in previous studies for vRNA labeling and dimerization [This report is the first in vivo visualization of the role of the DIS sequence in genome dimerization using a full-length RSV genome. The sequences required for dimerization in the RSV leader sequence were previously identified using in vitro dimerization assays with truncated vRNAs that contained the 5′ UTR and minimal sequences in the g region ,60,61,62g region . This fiackaging . It is urization ,47,48,76As mentioned previously, the RSV psi packaging sequence is located upstream of the 5′ splice acceptor site and is thus present on both spliced and unspliced vRNAs. RSV unspliced vRNA is preferentially packaged into newly formed virions, and studies have suggested that the genome dimerization is linked to genome packaging ,5,8,36. We applied our imaging techniques to characterize the genome composition of genome heterodimers within cells when either only unspliced vRNAs or both spliced and unspliced vRNAs were labeled with different fluorescent markers . We founOur studies also demonstrated that each imaging technique has unique characteristics and caveats. As previously noted, the smFISH technique resulted in detection of brighter RNA foci in the nucleus, likely due to accumulation of multiple copies of genomic vRNAs, and fainter RNA foci in the cytoplasm and at the plasma membrane, likely due to the presence of fewer copies of genomic vRNAs. The difference in intensity resulted in uneven detection by the Imaris Spots algorithm, which uses an intensity threshold to identify foci. This is likely the explanation for the decreased number of total colocalized red-green foci detected by smFISH-smFISH as compared to MS2-YFP-smFISH and MS2-YFP-Bgl-YFP . The use of MS2 and Bgl aptamer labeling systems resulted in higher levels of nonspecific background fluorescence due to the presence of fluorophore-tagged coat proteins that were not bound to vRNA. We also noticed a labeling bias between the MS2 and Bgl aptamers, with more Bgl-labeled foci visualized as compared to MS2-labeled foci intracellularly and within virions. The bias was not due to the nature of the fluorophore fused to the coat protein because when the fluorophores were swapped, a Bgl labeling bias was still seen . All of In summary, the results of this study demonstrate that RSV genomic RNA dimer formation occurs in the nucleus, and dimers may also arise in the cytoplasm and at the plasma membrane. Genome heterodimer formation is dependent on the presence of the L3 stem-loop and palindromic DIS sequence and appear to be composed of a pair of unspliced vRNAs. Additionally, we demonstrated the use of three distinct imaging approaches to visualize distinct single molecule RNA genomes in cells. The presence of genome dimers in the nucleus is intriguing considering that RSV ribonucleoprotein complexes arise in the nucleus and nuclear Gag is important for efficient packaging of genomic RNA dimers ,64,65,67


Estimates of changes in the frequency or height of contemporary extreme sea levels (ESLs) under various climate change scenarios are often used by climate and sea level scientists to help communicate the physical basis for societal concern regarding sea level rise. Changes in ESLs  are often represented using various metrics and indicators that, when anchored to salient impacts on human systems and the natural environment, provide useful information to policy makers, stakeholders, and the general public. While changes in hazards are often anchored to impacts at local scales, aggregate global summary metrics generally lack the context of local exposure and vulnerability that facilitates translating hazards into impacts. Contextualizing changes in hazards is also needed when communicating the timing of when projected ESL frequencies cross critical thresholds, such as the year in which ESLs higher than the design height benchmark of protective infrastructure  are expected to occur within the lifetime of that infrastructure. We present specific examples demonstrating the need for such contextualization using a simple flood exposure model, local sea level rise projections, and population exposure estimates for 414 global cities. We suggest regional and global climate assessment reports integrate global, regional, and local perspectives on coastal risk to address hazard, vulnerability and exposure simultaneously.The online version contains supplementary material available at 10.1007/s10584-021-03288-6. Extreme sea levels (ESLs) are short-lived (hours to days), exceptionally high local sea-surface heights, usually resulting from coastal storms, waves, or astronomical tides , nor do they consider vulnerability. Some human settlements may be protected to a level above the elevation of the ESL in question , or there may exist little to no exposure at or below the ESL height . Locally, ESLs AFs can be anchored to salient impacts on human systems and the natural environment, such as the frequency of overtopping existing flood defenses, roadway flooding, sewer or drainage back-ups, and the depth of the historically experienced high spring tide , will occur annually or more frequently at most available locations for RCP8.5 by the end of the century (high confidence)” . For example, by 2070, the frequency of the contemporary 100-year ESL for San Juan (Puerto Rico) is projected to increase from 0.01 events/year to > 31 events/year, on average, under a scenario in which global average surface air temperature (GSAT) stabilizes at + 2 ∘C GSAT stabilization scenario  is expected to at least double the 2010 population residing on lands below the 100-year ESL elevation for 25% of the 414 coastal cities assessed, growing to 37% under an end-of-century + 5 ∘C GSAT scenario.”To illustrate this discrepancy globally, we consider both the expected change in the frequency of the contemporary 100-year ESL at tide gauges matched to 414 global cities . However, also of interest is analyzing projection uncertainty in the rates of RSLC and how it impacts the timing of reaching ESL frequency benchmarks, such as the first year in which the contemporary 100-year ESL becomes the 1-year ESL. Just as with assessing ESL AFs for particular years, impactful communication of the timing of such benchmarks should be tied to relevant societal thresholds, rather than arbitrary water levels. Examples include the year in which ESLs higher than the design height benchmark of protective infrastructure  are expected to occur within the lifetime of that infrastructure  is expected to at least double the 2010 population residing on lands at or below the elevation of the 100-year ESL before 2100 at 27% of the 414 coastal cities assessed. This fraction grows to 54% under an end-of-century + 5 ∘C GSAT scenario.” We note that considering population exposure produces drastically different summary statements from those given in SROCC that only consider the hazard, for example stating that “most” locations will experience the contemporary 100-year ESL annually by 2100 . The focus on a doubling of population exposure is an arbitrary example; other societal thresholds could be explored.While these analyses are local, they can be aggregated to regional or global scales to better inform aggregate statements about uncertainty in the timing of changes in ESLs. For example, using our analysis, a summary statement could be constructed that communicates uncertainty in the timing of population exposure doubling, “projected 2070 RSLC under an end-of-century + 2 Extreme sea level (ESL) frequency amplification factors (AFs) are easy-to-calculate metrics that can help communicate flood and sea level rise risks when anchored to salient local events, such as the flooding of roadways, property, and other infrastructure , it is likely to be challenging (if not impossible) to implement in the IPCC’s main assessment reports . This is because physical projections are separated from societal impacts by design, the former appearing in the Working Group 1 (WGI) report, ahead of societal impacts that are released roughly 6 months later in Working Group 2 (WGII). Because of this separation, changes in hazards are inherently provided without context until the WGII report is released. In the interim period between these two reports, scientists, the media, and the public are forced to make subjective judgements about the impacts of these hazard changes without their explicit quantification. Potential solutions include tighter integration among working groups in future assessment cycles  or using Special Reports more often, the chapters of which are often more integrated than of those from the main assessment reports .New and existing datasets of terrain and flood protection elevation can help meet the needs of global assessment reports that seek to contextualize hazards. However, if there are gaps in the literature regarding these data, assessment reports are unlikely to fill the need on their own. Some existing datasets could help. The continually updated Dynamic and Interactive Vulnerability Assessment database (DIVA) is a popular source of vulnerability and exposure data for global-scale coastal flood risk assessments . New near-global-scale DEMs have provided increased accuracy for population exposure assessments  are protected by flood protection such as levees, seawalls, and deliberately raised structures


Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase—α, β, β′. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β’ subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.RNA turnover is essential in all domains of life. The endonuclease RNase Y ( Escherichia coli and Bacillus subtilis have generation times of 20 to 30 min. On the other hand, bacteria are exposed to a variety of changing environmental conditions, and due to their small size, the impact of environmental changes is particularly severe for bacterial cells. To adapt to these potentially rapidly changing conditions, bacteria have evolved a huge arsenal of systems to sense and respond to the environment. Especially in the competition between microorganisms, it is crucial that these responses are both rapid and productive. However, while regulatory events may be very rapid, there is an element of retardation in the system, and this is the stability of mRNA and protein molecules. If the continued activity of a protein may become harmful to the bacteria, it is important not only to prevent expression of the corresponding gene but also to take two important measures: (i) switch off the protein's activity and (ii) degrade the mRNA to exclude further production of the protein. The inactivation or even degradation of proteins is well documented in the model bacteria. For example, in both E. coli and B. subtilis the uptake of toxic ammonium is limited by a regulatory interaction of the ammonium transporter with GlnK, a regulatory protein of the PII family . In this study, we have addressed the essentiality of RNase Y in B. subtilis strains used in this study are listed in Table trpC2). B. subtilis and E. coli cells were grown in Lysogeny Broth (LB medium) (All  medium) . LB plat medium) . The plaB. subtilis was transformed with plasmids, genomic DNA or PCR products according to the two-step protocol . Illumina shotgun libraries were prepared using the Nextera XT DNA Sample Preparation Kit and subsequently sequenced on a MiSeq system with the reagent kit v3 with 600 cycles  as recommended by the manufacturer. The reads were mapped on the reference genome of B. subtilis 168 (GenBank accession number: NC_000964)  . Mapping_000964) . Frequen_000964)  and sile_000964) ,45.rny, rpoA, and cspD genes was achieved by transformation with PCR products constructed using oligonucleotides to amplify DNA fragments flanking the target genes and intervening antibiotic resistance cassettes as described previously  containing Mycobacterium smegmatis RNA polymerase core subunit genes was used as a template to create an analogous vector containing the genes rpoA, rpoZ, rpoE, rpoY and rpoBC. The construct was designed to allow removal/substitution of each gene via unique restriction sites  via XbaI and NotI restriction sites. The rpoB and rpoC genes were amplified by PCR using genomic DNA of B. subtilis 168 as a template and inserted into the plasmid via NotI and NcoI or NcoI and KpnI restriction sites, respectively. The rpoC gene was inserted with a sequence encoding a 8xHis tag on the 3′ end. The cloned construct was verified by DNA sequencing. The final vector, pBSURNAP, encodes a polycistronic transcript for expression of all six RNA polymerase core subunits. Expression is driven from an IPTG-inducible T7 RNAP-dependent promoter. Each gene is preceded by a Shine-Dalgarno sequence (AGGAG) except for rpoC. RpoB-RpoC are expressed as one fused protein connected by a short linker (nine amino acid residues) to decrease the possibility that E. coli subunits would mix with B. subtilis subunits as done previously for RNA polymerase from Mycobacterium bovis  and pGP2182 (RpoB-G1054C).To facilitate the purification of different variants of on sites . DNA encum bovis . The fulE. coli BL21 carrying pBSURNAP or the plasmids specifying the mutant alleles was cultivated in LB medium containing kanamycin (50 μg/ml). Expression was induced by the addition of IPTG  to logarithmically growing cultures (OD600 between 0.6 and 0.8), and cultivation was continued for 3 h. Cells were harvested and the pellets from 1 l of culture medium were washed in 50 ml buffer P  and the pellets were resuspended in 30 ml of the same buffer. Cells were lysed using a HTU DIGI-F Press . After lysis, the crude extracts were centrifuged at 41 000 × g for 30 min at 4°C, and the RNA polymerase was purified from the supernatant via the His-tagged RpoC as described  for 10 min at 30°C. Assays were carried out in 10 μl with 64 nM RNA polymerase holoenzyme and 100 ng plasmid DNA templates in transcription buffer containing 40 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.1 mg/ml bovine serum albumin (BSA), 150 mM NaCl, and NTPs . The samples were preheated for 10 min at 37°C. The reaction was started by the addition of RNA polymerase and allowed to proceed for 20 min (30 min in the case of iNTP-sensing experiments) at 37°C. Subsequently, the reaction was stopped by the addition of 10 μl of formamide stop solution . The samples were loaded onto 7 M urea–7% polyacrylamide gels. The gels were dried and exposed to Fuji MS phosphor storage screens, scanned with a Molecular Imager FX (BIORAD) and analyzed with Quantity One program (BIORAD).Multiple round transcription assays were performed as described previously , unless 600 of 0.5 to 0.6. 5 ml samples of the cultures were added to 10 ml RNA-protect (Qiagen) and allowed to incubate for 5 min at room temperature, followed by centrifugation at 5000 × g for 10 min at 4°C. Pellets were quickly frozen in liquid nitrogen and stored at −80°C. A total of three independent biological replicates were included. The harvested pellets were resuspended in 800 μl RLT buffer  with β-mercaptoethanol (10 μl/ml) and cell lysis was performed using a laboratory ball mill. Subsequently 400 μl RLT buffer with β-mercaptoethanol (10 μl/ml) and 1200 μl 96% [v/v] ethanol were added. For RNA isolation, the RNeasy Mini Kit (Qiagen) was used as recommended by the manufacturer, but instead of RW1 buffer RWT buffer (Qiagen) was used to facilitate the isolation of RNAs smaller 200 nt. To determine the RNA integrity number (RIN) the isolated RNA was run on an Agilent Bioanalyzer 2100 using an Agilent RNA 6000 Nano Kit as recommended by the manufacturer . Remaining genomic DNA was removed by digesting with TURBO DNase . The Pan-Prokaryozes riboPOOL kit v1  was used to reduce the amount of rRNA-derived sequences. For sequencing, the strand-specific cDNA libraries were constructed with a NEBNext Ultra II directional RNA library preparation kit for Illumina . To assess quality and size of the libraries, samples were run on an Agilent Bioanalyzer 2100 using an Agilent High Sensitivity DNA Kit . Concentration of the libraries were determined using the Qubit® dsDNA HS Assay Kit as recommended by the manufacturer . Sequencing was performed by using the HiSeq4000 instrument  using the HiSeq 3000/4000 SR Cluster Kit for cluster generation and the HiSeq 3000/4000 SBS Kit  were considered differentially expressed. The raw reads have been deposited in the National Center for Biotechnology Information's (NCBI) Sequence Read Archive (SRA) under accession no. SRP274247. Functional and regulation information on the differentially expressed genes was obtained from the SubtiWiki database . However, we observed the appearance of suppressor mutants after a few days. By analysis of such mutants we wished to gain a better understanding of the growth-limiting problem of the rny mutant. For this purpose, we isolated suppressor mutants in different experimental setups. First, the rny mutant GP2501 was adapted to growth in liquid LB medium at 22°C since the rny mutants had a severe growth defect at low temperatuRes. After the adaptation experiment, the culture was plated at 22°C, and two colonies were isolated for further investigation. In addition to the adaptation experiment in liquid medium, we also evolved suppressors on solid LB agar plates both at 22°C and 37°C. We isolated two mutants under each condition  in the n factor . For the25L in thrpoE gene encoding the δ subunit of RNA polymerase, which resulted in a frameshift after residue G66  had a deletion of the skin element, an amino acid substitution (Y55N) in the AdeR activator protein , and a sidue G66 ,62. For cspD gene encoding an RNA binding protein which has transcription antitermination activity in E. coli .The suppressor evolved at 37°C on LB plates GP2636) contained a mutation resulting in the introduction of a premature stop at the eighth codon of the 6 contain E. coli . Sanger ctsR-pdaB genomic region was accompanied by specific additional suppressor mutations affecting transcription in every single suppressor mutant analysed. These mutations result in the inactivation of the greA gene in liquid medium at 22°C, whereas the selective pressure on agar plates at 22°C and 37°C was directed at the inactivation of the RNA polymerase subunit RpoE or the RNA binding protein CspD, respectively . In the case of the rny cspD double mutant GP2615, complete suppression was observed  with at least two-fold differential expression in the Δrny strain GP2501 as compared to the wild type 168. It should be noted that the number of differentially expressed genes is likely to be underestimated, since about 50% of all genes are not or only very poorly expressed during vegetative growth  was observed for the yxkC gene. This gene codes for protein of unknown function and is part of the σD regulon  this reduced expression of target genes might cause the disordered cell wall of the rny deletion strain  are essential, and only the expression of ywpB encoding an enzyme of fatty acid biosynthesis is 2.4-fold reduced in the rny mutant. This weak regulation suggests that fatty acid biosynthesis is not the growth-limiting factor for the rny mutant. In contrast, many of these genes with  restored expression belong to prophage PBSX or are required for rather specific metabolic pathways. In conclusion, the evaluation of the genes which had their expression restored as a result of the suppressing mutations did not give a clear clue to the reason of suppression.Importantly, we wanted to test whether the suppressor mutant had restored a wild type-like expression of genes that were affected by the loss of RNase Y. We found 461 genes with differential expression between the suppressor mutant GP2518 and the 2518 see . Among trny deletion. Therefore, we hypothesized that the duplication of the genes encoding the main three subunits of RNA polymerase made a major contribution to the selective advantage provided by the duplication.As mentioned above, the region duplicated in all suppressor mutants contained genes encoding RNA modification enzymes, translation factors, ribosomal proteins, RNases, and proteins involved in transcription. MrnC and Rae1 are RNase Mini-III required for the maturation of 23S rRNA and ribosome-associated A site endoribonuclease, respectively ,69. As orrn gene clusters which may facilitate the duplication event .To test the idea that simultaneous duplication of all three genes for the RNA polymerase core subunits is key for the suppression of the loss of RNase Y, we decided to interfere with this possibility. The duplicated region is located between two highly conserved rny gene, and to isolate suppressor mutants. Indeed, even with the genomically separated RNA polymerase genes, suppressor mutations appeared upon the deletion of the rny gene encoding RNase Y. There were three possibilities for the outcome of the experiment. First, the same genomic region as in the original suppressors might duplicate thus falsifying our hypothesis that the simultaneous duplication of all three genes encoding the core subunits of RNA polymerase is required for suppression. Second, both regions containing the rpoA and rpoBC genes might be duplicated. Third, in the new genetic background completely new suppressing mutations might evolve. Two of these suppressor mutants were subjected to whole genome sequencing. None of them had the duplication of the ctsR-pdaB region as in the original suppressors. Similarly, none of the mutants had the two regions containing the rpoA and the rpoBC genes duplicated. Instead, both mutants had point mutations in the RNA polymerase subunit genes that resulted in amino acid substitutions  into the wild type background of B. subtilis 168. For this purpose, the CRISPR/Cas9 system designed for use in B. subtilis was employed  in rae1 (strain GP2901), we failed to isolate genome-edited clones expressing the RpoC-R88H variant in multiple attempts. This failure to construct the RpoC-R88H variant in the wild type background suggests that the properties of the protein are altered in a way that is incompatible with the presence of an intact RNA degradation machine.Based on the essentiality of transcription, we expected that the mutations in employed . As a coin vivo failed, we decided to test the properties of the mutant RNA polymerases using in vitro transcription. B. subtilis RNA polymerase is usually purified from a strain expressing His-tagged RpoC  and pGP2182 (RpoB-G1054C) . The variant RNA polymerases were expressed in E. coli BL21 and purified via affinity chromatography and subsequent size exclusion chromatography.Since our attempts to study the effect of the mutations ged RpoC . Howevermegmatis . Brieflyin vitro transcription on three different templates, containing well-studied promoters of the veg and ilvB genes and the P1 promoter of the rrnB operon (We purified the wild type and two mutant RNA polymerases (RpoC-R88H and RpoB-G1054C) and assessed their activity by B operon ,74. In aB operon , this enB operon .rrnB operon, B. subtilis RNA polymerase is sensitive to the concentration of the first transcribed nucleotide both in vitro and in vivo  in addition to a duplication of the chromosomal region encoding the core subunits of RNA polymerase. The second class had point mutations affecting the β or β’ subunits of RNA polymerase that result in strongly decreased transcription activity. At a first glance, these results seem to be conflicting. Considering RNA degradation as the function of RNase Y, it seemed plausible that the selective pressure caused by deletion of rny would result in alleviating the stress from mRNA accumulation. This seems to be the case in the second class of suppressors (see above), whereas the reason for the duplication seems to be less obvious. Importantly, this duplication was always accompanied by one of the other aforementioned mutations reducing transcription efficiency. In an attempt to determine the order of the evolutionary events in these suppressors we established a method to detect the presence of the duplication without whole genome sequencing. For this, we made use of a pair of oligonucleotides that binds to the pdaB and ctsR genes giving a product of about 10 kb, if the region is duplicated or amplified but no product in the absence of duplication or amplification  and therefore only detectable by the very sensitive PCR assay.It is well-established that genomic duplications or amplifications occur frequently in bacterial populations, even in the absence of selective pressure . In Salmications . Since e regions ,45. Inderny mutants generate distinct selective forces: While the duplication is not fixed in strains with separated rpo genes, it seems to become fixed in the suppressor mutants that have the rpo genes in one genomic region. To investigate the order of evolutionary events, we cultivated the rny mutant strain GP2501 for 75 h and monitored the status of the rpoA-rpoBC chromosomal region by PCR  to whole genome sequencing. The sequencing confirmed presence of the duplication, but did not reveal any additional suppressor mutation. Based on this result, we can assume that upon deletion of rny the bacteria first fixed the duplication of the pdaB-ctsR region and then, later, may acquire the point mutations affecting greA, rpoE, or cspD.Obviously, the different genomic and genetic backgrounds of the rny deletion in strains with one or two copies of the rpoA, rpoB, and rpoC genes. For the duplication of the core subunit genes, we first assembled a second chromosomal copy of the rpoBC operon adjacent to the purT gene . Into the resulting strain GP3288, we inserted the second copy of the rpoA gene and then deleted the rny gene  supporting the broad relevance of these two enzymes  that may affect the mRNAs of essential genes as has been found for B. subtilis RNase III and E. coli RNase P  and point mutations in greA, rpoE, and cspD that all affect transcription. If this duplication was prevented by genomically separating the RNA polymerase genes, we found suppressor mutants affecting the core subunits of RNA polymerase which result in strongly compromised transcription activity. Taken together, these findings suggest that the (quasi)-essentiality of RNases E and Y is related to their general function in initiating mRNA turnover rather than to the processing of specific RNA species. This idea is further supported by three lines of evidence: First, mutations that mimic a stringent response and therefore reduce RNA polymerase activity suppressed the growth defect of a rne mutant. Second, artificial expression of RNase Y or of the ribonucleases RNase J1 or J2 from B. subtilis partially suppressed the E. coli strain lacking RNase E, but only under specific growth conditions , and third, RNase E can functionally replace RNase Y (A lot of effort has been devoted to the understanding of the reason(s) of the (quasi)-essentiality of RNases E and Y for  RNase P ,79,80. Hectively ,82. In aectively . Yet anoe mutant . The res RNase Y .E. coli and B. subtilis, respectively, one might expect that the overexpression of other RNases might compensate for their loss. By analogy, such a compensation has been observed for the essential DNA topoisomerase I of B. subtilis, which could be replaced by overexpression of topoisomerase IV -essential function of RNases E and Y in erase IV . Howevererase IV , we nevee mutant ,88 suggepdaB-ctsR chromosomal region are required to compensate for the loss of RNase Y.An interesting result of this study was the apparent contradiction between the isolation of suppressor mutants with increased copy number of core RNA polymerase subunit genes in one setup, intuitively suggesting increased transcription activity, and the isolation of mutants that exhibited severely reduced RNA polymerase activity in the other setup. We therefore tested with a theoretical model whether duplication of the core subunits leads to abortive incomplete complexes, as the composition of the RNA polymerase complex might be perturbed by the duplication of the core. The model confirmed that perturbing the stoichiometry of the transcription machinery may result in a strong reduction of the fraction of core RNA polymerases that assemble a functional complex. However, our experimental data demonstrate that duplication of the RNA polymerase core genes is not sufficient for suppression indicating that other factors encoded in the B. subtilis is accompanied by the acquisition of mutations that strongly reduce transcription activity of the RNA polymerase. In fact, the reduction of activity was so strong that it was not tolerated in a wild type strain with normal RNA degradation. This indicates that the suppressor mutants have reached a new stable equilibrium between RNA synthesis and degradation, which, however, is not optimal as judged from the reduced growth rates of the suppressor mutants as compared to the wild type strain. It has already been noticed that generation times and RNA stability are directly related (B. subtilis and Mycobacterium tuberculosis might be a factor to achieve this coupling between RNA synthesis and degradation (In each organism, an optimal trade-off between RNA synthesis and degradation must be adjusted to allow optimal growth. Obviously, the loss of the major RNA decay-initiating enzyme will bring this adjustment out of equilibrium. This idea is supported by the observation that reduced RNA degradation in  related ,89. Thisradation ,91.B. subtilis. In addition to RNase Y, RNase J1 is also quasi-essential for this bacterium. In the future, it will be interesting to understand the reasons behind the critical role of this enzyme as well in order to get a more comprehensive picture of the physiology of RNA metabolism.In conclusion, our study suggests that the initiation of mRNA degradation to keep the equilibrium between RNA synthesis and degradation is the function of RNase Y that makes it quasi-essential for gkab528_Supplemental_FileClick here for additional data file.