Regulation of gene expression in any biological system is a complex process with many checkpoints at the transcriptional, post-transcriptional and translational levels. The control mechanism is mediated by various protein factors, secondary metabolites and a newly included regulatory member, i.e., noncoding RNAs (ncRNAs). It is known that ncRNAs modulate the mRNA or protein profiles of the cell depending on the degree of complementary and context of the microenvironment. In plants, ncRNAs are essential for growth and development in normal conditions by controlling various gene expressions and have emerged as a key player to guard plants during adverse conditions. In order to have smooth functioning of the plants under any environmental pressure, two very important DNA-harboring semi-autonomous organelles, namely, chloroplasts and mitochondria, are considered as main players. These organelles conduct the most crucial metabolic pathways that are required to maintain cell homeostasis. Thus, it is imperative to explore and envisage the molecular machineries responsible for gene regulation within the organelles and their coordination with nuclear transcripts. Therefore, the present review mainly focuses on ncRNAs origination and their gene regulation in chloroplasts and plant mitochondria. Technology improvements in the high-throughput sequencing of DNA and RNA and subsequent numbers of studies have provided extensive information on ncRNAs and their complex regulation networks in biological systems. In plants, more than 80–90% of transcripts are non-protein-coding RNAs ,2, whereArabidopsis [Generally based upon length, ncRNAs are categorized into three groups: small ncRNAs (sRNAs) with 18–30 nucleotides (nt) length, medium-sized ncRNAs (31–200 nt) and long ncRNAs (lncRNAs) (>200 nt). Further, based upon their origins, processing and mode of action, small ncRNAs are mainly divided into two classes, such as small interfering RNAs (siRNAs) and microRNAs  ,5. In thbidopsis , soybeanbidopsis  and cucubidopsis ,12,13,14Although recent advancements in genomic and transcriptomic analyses have enabled researchers to trace the presence of chloroplast-originated ncRNAs , only foChloroplasts are a kind of a membrane-bound cell organelle, embedded within the outer and inner membranes. They range in size from 5 to 10 µm with highly variable shapes, existing as cup-shaped, spiral-shaped or star-shaped forms in algae, whereas they are saucer-shaped in bryophytes and higher plants . The outrpoA , clpP (ATP-dependent caseinolytic protease) and ndhD (subunit of NA(P)DH dehydrogenase (NDH) complex) [WSL” (white stripe leaf) which codes for one chloroplast-localized PPR that is imperative for splicing of a chloroplast transcript, rpl 2 . To substantiate WSL essentiality in plants, the mutation in WSL leads to sensitivity towards several stress responses. Similarly, apart from PPR, many other RBPs such as RLSB, an S-1 domain RNA-binding protein, and chloroplast ribonucleoprotein 31A are involved in regulation of organelle gene expression [It has been estimated that one cell can contain ~10,000 copies of plastid DNA, attached to the thylakoid membrane along with the proteins and RNAs which arenamed nucleoids ,29. A plcomplex) . Tan et complex)  exploredpression ,40.Plant development and stress tolerance capability are vastly determined by the photosynthetic yield occurring in the green organelle. Therefore, it is necessary to have a proper orchestral coordination between the nucleus and chloroplasts for unperturbed functioning of the organellar metabolic processes and, hence, the plant system. Besides carrying out photosynthesis, chloroplasts are crucial to conduct many other metabolic processes such as nitrogen and sulfur assimilation and nucleotides, amino acids and fatty acids synthesis. In addition, the green organelle also generates different kinds of secondary metabolites which act as signaling effectors during growth and development or in any stress condition . Most of+ oxidoreductase (phosphorylating) [Zanthoxylum bungeanum (a shrub from the Rutaceae family) were found to be related to chlorophyll accumulation, chloroplast stability and photosynthesis. These nuclear-encoded lncRNAs were observed to be precursors for many miRNAs that control the expression of several chloroplast-associated targets such as PHOT2(Phototropin-2), ARC3(Protein accumulation and replication of chloroplast 3) and UGP3(UTP glucose-1-phosphate uridylyltransferase 3), among which ARC3 and UGP3 code for chloroplast-localized proteins [Chloroplasts regulate the expression of nuclear genes via various plastid-originated signaling molecules, called “retrograde signaling”. Such signaling communication mostly takes place in response to developmental events or other external factors such as abiotic and biotic stress conditions that directly have an effect on photosynthesis. Chloroplast-to-nucleus signaling was first observed in barley mutant lines by measuring the reduction in nuclear genome-encoded plastid enzymes: phosphoribulokinase and D-glyceraldehyde-3-phosphate: NADPylating) . These rylating) . Later, ylating) . Severalylating) ,45. On tylating) ,47. Receproteins . In addiPervasive genome transcription occurring in chloroplasts causes random origins of transcription from genic and intergenic regions that leads to generation of unexpected transcriptional outputs . EventuaatpE. Afterwards, a study from sRNAs profiling under the tomato fruit ripening phase revealed a highly expressed cluster of sRNAs (named cluster “F”) in the chloroplast genome. However, the origin of sRNAs from “F” loci is yet to be deciphered due to the unannotated tomato genome which may have a part of the plastid genome via integration [Cucumis melo showed that around 30,000 candidates mapped with the chloroplast genome, among which two clusters were found to be located within rRNA genes both in sense and antisense orientations [A very first comprehensive report on ncRNAs from chloroplasts was provided by Lung et al.  by examiegration . Out of egration . Similaregration ,71,72. sntations . One of Nicotiana benthamiana [Arabidopsis thaliana, 19–26 nt-long plastid tRNA-derived ncRNAs (plastid tRNA fragments) were detected through sRNA deep sequencing. More than 50% of the plastid tRNA fragments (ptRFs) arise from the 5′ end of tRNA with higher accumulation in cold-stressed than control plants. Surprisingly, Northern blot analysis of nucleus, cytoplasmic and plastid RNA fractions showed that plastid tRNA-derived ncRNAs are located in the cytoplasm but not in chloroplasts [Interestingly, yet another report from high and moderate temperature-treated Chinese cabbage exhibited the presence of plenty of chloroplast-generated sRNAs with 9–36 nt length . The valthamiana , which rthamiana  predictethamiana  comparedthamiana ,75,76. Iroplasts , which iroplasts . It may roplasts ,71.Chlamydomonas reinhardtii, the functional link between a nucleus-encoded RNA maturation factor, MMB1 (a member of the PPR family), and small RNAs mapping to the 5′ UTR of psbB and psbH was examined. It was concluded that these sRNAs from algal chloroplasts could be important as a landmark for identification of cis-acting elements essential for mRNA accumulation [Amphidinium carteraei, showed the existence of noncoding sequences in the psbA and petB/atpA regions [psbA and petB/atpA regions from various reports based on the chloroplast transcriptome provides a preliminary platform to analyze their functional importance in photosynthetic process regulation [Apart from higher plant chloroplasts, ncRNAs have also been identified in lower divisions of the plant kingdom such as algae. In a green alga, mulation . Similarmulation . Strikinmulation . Recentl regions . RNA hybgulation ,80.clpP–rps12, atpH–atpF, cemA–petA, rpl18–rps20, rps7–ndhB, rps15 and psaI–ycf4 in Arabidopsis thaliana revealed that most of the sRNAs reside close to the 5′ terminus and few reside at the 3′ end of mRNAs. It should be highlighted that these sRNA were highly conserved among the monocot and dicot plant chloroplast genome [Arabidopsis thaliana) displayed absence of its respective 11 sRNAs [Arabidopsis were located in intergenic regions and few were from coding sequences and intron regions. Interestingly, a large number of termini belonged to an sRNA that was a footprint of PPR proteins. Furthermore, the sequencing analysis showed the importance of the 3′ processing enzyme chloroplast polynucleotide phosphorylase (cpPNPase) in plastid RNA processing as they registered an increased number of altered termini in a pnp 1-1 mutant plant [Arabidopsis thaliana chloroplast transcriptome analysis carried out previously by Hotto and coworkers [In spite of having the above-mentioned evidence on chloroplasts rRNAs from several transcriptome analyses in plants, many studies have proposed most of these organellar ncRNAs as a footprint of PPR proteins, RNA-binding proteins protecting plastid mRNAs’ termini from exonucleases ,81,82. At genome . An RNas11 sRNAs  used Ternt plant . Similaroworkers  revealedArabidopsis thaliana, Nicotianatabacum and poplar, which covered the RNA editing site and group II intron splice acceptor site. The length of this antisense RNA varied from 400 to 650 nt and showed varied expression under different developmental stages. It was anticipated that asRNA_ndhB might be involved in transcript maturation or RNA stability. Interestingly, ectopic expression of chloroplast antisense RNA “AS5” in N. tabacum showed many folds of accumulation of the related sense transcript tRNAArg than 5S rRNA. Additionally, a reduction in ribosomal association of 5S rRNA and rbcL mRNA was observed, suggesting that AS5 may be needed for stability. Therefore, it was explained that this asRNA might play an important regulatory role in translation by controlling the 5S rRNA level [Arabidopsis. Thus, it can be hypothesized that AS5 might be involved in inhibition of 5S rRNA maturation through endonucleolytic cleavage of precursor 5S rRNA by an RNaseIII-like enzyme [S. Miltiorrhiza and a number of chloroplasts-related lncRNAs from V. venifera were reported [The length of organellar ncRNAs has a broad range. The sRNAs reported from chloroplasts can be lengthier than nuclear genome-transcribed sRNAs  or, in sNA level . To subse enzyme . Soon afreported ,87. GiveMuch like chloroplasts, mitochondria have their own genome and this semi-autonomous organelle originated from the symbiosis of an alpha-proteobacterium with a proto-eukaryotic host ,89. Mitotrans-elements: PPR, RBPs, DEAD-box RNA helicases and mitochondrial transcription termination factors (mTERF) [Similar to chloroplast DNA, mitochondrial DNA transcription appears to be a complex process and post-transcriptional events are governing factors in organelle gene regulation that mostly depends on inter-organelle coordination. RNA metabolism is mostly controlled by nuclear-encoded RBPs ,90,103.  (mTERF) ,107,108. (mTERF) , Dietric (mTERF) ). In man (mTERF) ,109,110.Silene noctiflora revealed 97 editing sites outside of the annotated sequences, which evidenced that noncoding transcripts coming from outside of genic regions are being processed by editing factors [Arabidopsis thaliana mitochondria have been identified using sRNA sequencing data and the bioinformatics pipeline “sRNA miner” [Studies on plant mitochondrial ncRNAs as regulatory molecules are ata naïve stage. Previously, a review by Dietrich et al.  on ncRNA factors . Further factors . AnalogoA miner” . ResearcA miner” . In addiA miner” . Silene vulgaris explored an 18 nt in length noncoding region from the transcriptome of floral bud tissue from a female plant (F) and hermaphrodites (H), named “Female Specific Noncoding RNA” (FSNR) [Another study on cytoplasmic male sterility (CMS)-related genes in ” (FSNR) . This wa” (FSNR) ,115,116.” (FSNR) . Moreove6A modifications in mitochondrial intergenic noncoding transcripts from Arabidopsis thaliana and Brassica oleracea. The common sequence motifs analysis revealed a transcript with m6A modifications, NNHRm6AYSNN , which shows similarity with the m6A motif present on nuclear-encoded mRNAs [6A methylation modification was observed to happen in this region [6A modifications in ncRNAs may eventually affect their cognate targets. However, in this perspective, there are no proper reports available on regulatory roles of plant mitochondrial ncRNAs on target genes. Circular RNAs (circRNA), which are a type of long ncRNA, have been revealed as new emerging regulatory molecules in biological systems [N. benthamiana, where these were found to be involved in the regulation of the mitochondrial genome [The gene regulation through nucleic acid base modifications and its impact on plant development and tissue differentiation have been documented in various studies . Interesed mRNAs . Hence, s region . Therefo systems ,121,122.l genome .It is well known from several elaborative studies that animal mitochondrial ncRNAs involved in retrograde or anterograde signaling can play important roles in various gene regulatory networks. Therefore, it can be presumed that ncRNAs associated with plant mitochondria may also have such roles in plant systems . Given tRecent searches from RNA sequencing data have proved that organelle DNA transcription is pervasive ,127, givArabidopsis. It was postulated that these antisense RNAs might be crucial for suppressing NEP-generated mRNAs during subsequent developmental stages where PEP activity becomes significant [Chlamydomonas chloroplasts, though few genes are cotranscribed, each individual gene also has its own promoter which might give rise to a cosRNAs TSS [Silene vulgaris, was speculated to have its own promoter as it was not overlapping with any other transcribed region [High activity of NEP in the plastid during germination was suggested to cause complete transcription that leads to an elevated amount of antisense RNAs production in nificant . Interesnificant . The accRNAs TSS , as notiRNAs TSS . Recentld region . Thus, nArabidopsis suggested that there can be several different routes for ncRNAs biogenesis and degradation. CpPNPase is among one of the RNases that contribute in organelle RNA metabolism [Arabidopsis [It is well known that ribonucleases and other RBPs play important roles in organellar RNA quality control. RBPs can possibly be leading a parallel pathway for regulatory sRNAs generation or their degradation if they are not required by an organelle. A chloroplast RNase mutation study in tabolism . Changestabolism . Anothertabolism ,128. Thebidopsis . Thus, tPPR proteins in chloroplasts and mitochondria are known as RNA protectors. They protect 5′ or 3′ ends of RNAs from exonucleolytic degradation caused by RNases, which are speculated to produce PPR footprints ,130. On Interestingly, Gomez and Pallas  studied With the recent progress in genome and transcriptome analysis, today, we have a large collection of genomic data for eukaryotic and prokaryotic organisms from different habitats. NGS technology has played a significant role in creating a huge depository of transcriptomic data related to protein coding transcripts or regulatory noncoding transcripts.PEP mutants, exhibited numerous TSSs on antisense strands or in intergenic regions of the plastome [Arabidopsis thaliana also found numerous transcription start sites in intergenic regions, pointing towards the existence of several ncRNA promoters [Mapping of the TSS on the plastid genome could serve as one useful method to predict and assert the presence of ncRNAs in chloroplasts. A novel differential RNA sequencing approach for plastids, isolated from plastome . Recentlromoters . HoweverIn combination with high-throughput sequencing of theorganelle genome and transcriptome, computational analyses based on certain criteria have also been considered as significant tools to recognize functional organellar ncRNAs. However, the accuracy of prediction of ncRNAs based upon any algorithm typically depends on the information parameters chosen during analysis. Wu et al.  recentlyVal that was developed from the 3′ end of Turnip yellow mosaic virus (TYMV) genomic RNA), was demonstrated for serving the purpose of passenger RNA (ribozyme Rzatp9) translocation from the cytoplasm to mitochondria in tobacco cells and the Arabidopsis thaliana plant [Another approach to analyze the functional importance of any transcript in mitochondria and chloroplasts could be to knockdown the organellar transcript by transporting a customized RNA inside the organelle. In various studies, it has been shown that tRNA can carry a passenger RNA molecule to mitochondria ,140. In na plant ,142. In Today, we have various techniques to study ncRNA-mediated gene regulation in multicellular organisms. Nonetheless we need to conduct a series of experiments in various aspects to validate the genuineness and functional importance of organelle-generated ncRNAs, as it is known that organelle genome fragments are also present in the nuclear genome. In addition, organelle gene expression is partially regulated by the nucleus. Therefore, conducting a comparative study by taking purified organellar RNA/DNA and total cellular RNA/DNA under various physiological conditions, as has been conducted previously in many studies, can be an appropriate method to gain an unambiguous picture. However, a signal-based RNA detection assay or RNA imaging could be a rapid method to trace organellar ncRNAs. Many researchers have recently explored the scope of utilizing the highly sensitive CRISPR -based tools in RNA detection or sub-cellular localization of RNA molecules . For an In this review, we have explained the current scenario of different noncoding RNA molecules with respect to their generation and functional potential in plant sub-cellular compartments. There are enormous reports available on nuclear genome-transcribed functional ncRNAs in different plant species stating their roles in regulation of growth and development and their involvement in regulation of stress-responsive factors. On the other side, limited research has been conducted on ncRNAs from two other plant cell organelles, i.e., chloroplasts and mitochondria. Over the past decade, a good number of transcriptomic studies from chloroplasts have shown the presence of enormous ncRNAs, though further validation is required. Several plastid-encoded asRNAs were proposed to play an important role either as a negative or positive regulator of sense strands. Additionally, several cosRNAs reported ranging from algae to higher plant chloroplasts and mitochondria are mapped with regulatory 5′ or 3′ UTRs of coding sequences, which shows their possible importance in post-transcriptional regulation of the related genes. In addition, ncRNAs accumulation seems to be dependent on different developmental stages of plants and environmental conditions.Relaxed transcription of the organelle genome generates many intergenic and antisense transcripts along with coding transcripts, and depending upon organellar metabolic activities, the noncoding transcripts are degraded by different nuclear-encoded ribonucleases. However, many RNA sequencing studies in normal and ribonuclease mutant plants have proposed the significance of ncRNAs in regulation of organelle genes. So far, it is established from previous studies that RNA metabolism in chloroplasts and mitochondria is mostly regulated by RBPs and ribonucleases; on account of actions by these two kinds of proteins, RNA footprints are produced which might mostly be destined to be degraded. Strikingly, the RNA fragments generated due to transcript protection action by the PPR protein were proposed as potent competitors of cognate mRNAs for binding with PPR; in other words, these may be one of the deciding factors for mRNA translation . Detecti


OCTs and OCTNs expression, along with that of Amino acid Transporter B0,+ transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway™, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side; there, the most abundant carrier is, instead, SLC6A14/ATB0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung.Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney; in the lung, OCTs mediate inhaled drugs’ transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines; here, we studied OCTs in EpiAirway™, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP Human transporters belonging to the solute carrier family 22A (SLC22A) play a central role in physiology, pharmacology, and toxicology because of the broad spectrum of endogenous metabolites, drugs, and toxins that they can move across cell plasma membranes ,2.+-cotransport of selected zwitterions, and Organic Anions Transporters (OATs), that physiologically work as organic anion exchangers MPP+ . Where indicated, the inhibitors or the drugs were added to the transport buffer at the indicated concentrations. For a sodium-free EBSS, 117 mM NaCl was replaced with equimolar N-methyl-d-glucamine chloride. At the times indicated, transport buffer was removed, and the experiment was terminated by two rapid washes (<10 s) in ice-cold urea (300 mM). The filter was then detached from the insert and the ethanol soluble pool was extracted from monolayers. Radioactivity in cell extracts was determined with MicroBeta2 liquid scintillation spectrometer . In order to discriminate the contribution of OCT1 and OCT3 transporters, MPP+ uptake was measured both in the absence and in the presence of quinidine or corticosterone employed as preferential inhibitors of OCT1 urea, as already described [14C]urea . The incubations were terminated by two rapid washes (<10 s) in ice-cold 300 mM urea and radioactivity in cell extracts was determined as described above. Calculated cell volumes corresponded to 5.2 ± 0.12 (for EpiAirway™) and 5.4 ± 0.6  μL/mg of protein (not shown).MPP of OCT1 ,19 and O of OCT1 , respect of OCT1 . MPP+ upescribed . BrieflymRNA expression has been analysed through RT-qPCR, as previously described . 1 µg ofeviously . cDNA obTo monitor the expression of transporter proteins, cell monolayers were washed with ice-cold PBS and lysed in Laemmli Sample Buffer , 20% glycerol, 0.2 M dithiothreitol (DTT)). Western Blot analysis was then performed as previously described , by emplt-test for unpaired data.The statistical analysis was performed using GraphPad Prism 7 (GraphPad Software). All data were analyzed with a two tailed Student’s N-Methyl-4-phenylpyridinium acetate-[N-methyl-3H] (MPP+), 81.3 Ci/mmol, and [14C]-Urea, 55.42 mCi/mmol, were obtained from Perkin Elmer. All other chemical and reagents were from Sigma-Aldrich. +) has been initially measured at different times, up to 30 min, at both sides of EpiAirway™  or of tumor necrosis factor α (TNFα). The results obtained, shown in SLC22A1/OCT1 nor SLC22A3/OCT3 in either cell model. We then checked the possibility that inflammatory stimuli may modify the expression of OCT transporters, making them potential targets of anti-inflammatory drugs. To this end, we evaluated the expression of +-Cl− cotransporter Amino acid Transporter B0,+  to carnitine uptake at the apical side. Therefore, in order to draw a complete picture of the expression of the transporters for organic cations in EpiAirway™, we quantified the amount of mRNA for OCT1 and OCT3 carriers, along with that of ATB0,+ and OCTN2  at the apical side of polarized Calu-3 cells is inhibited by bronchodilators formoterol, salbutamol, ipratropium, and the glucocorticoid budesonide [The role of OCTs and OCTNs in the uptake of drugs is, instead, well recognized, and their involvement in the intestinal absorption and renal excretion of many cationic drugs is well established ,5,33. Thdesonide . Consistdesonide , also in0,+. The latter could thus be potentially listed, along with OCTs and OCTNs, among organic cation transporters responsible for drug delivery in the lung.In conclusion, our results indicate that EpiAirway™ lack any luminal activity of OCTs and OCTNs, and OCT1 and OCT3 are, indeed, active on the basolateral membrane and not at the apical side, where the major carrier involved in the transport of organic cation solutes is ATB


C. elegans is secreted from the soma and taken-up by the germline to ensure germline immortality at elevated temperature. ENDU-2 binds to mature mRNAs and negatively regulates mRNA abundance both in the soma and the germline. While ENDU-2 promotes RNA decay in the soma directly via its endoribonuclease activity, ENDU-2 prevents misexpression of soma-specific genes in the germline and preserves germline immortality independent of its RNA-cleavage activity. In summary, our results suggest that the secreted RNase ENDU-2 regulates gene expression across tissue boundaries in response to temperature alterations and contributes to maintenance of stem cell immortality, probably via retaining a stem cell specific program of gene expression.Multicellular organisms coordinate tissue specific responses to environmental information via both cell-autonomous and non-autonomous mechanisms. In addition to secreted ligands, recent reports implicated release of small RNAs in regulating gene expression across tissue boundaries. Here, we show that the conserved poly-U specific endoribonuclease ENDU-2 in  C. elegans that the poly-U specific endoribonuclease ENDU-2 is secreted from the soma and taken-up by the germline, binding mature mRNA, repressing expression of somatic transcripts and maintaining germline immortality.The question as to how the soma regulates the germline has been much studied. Here, the authors show in  C. elegans, loss of germline immortality typically results in steadily increased incidence of sterility of initially fertile animals over generations. To date, telomerase-mediated telomere maintenance, balanced heterochromatic H3K9 methylation, and euchromatic H3K4/H3K36 methylation are the prevalent known molecular mechanisms involved in maintenance of germline immortality4. Piwi RNA mutants and mutations in multiple components of a nuclear RNA interference (RNAi) pathway that promotes the inheritance of germline RNAi also result in a temperature-dependent mortal germline (Mrt) phenotype6. Failure to maintain germline immortality in Piwi RNA mutants has recently been explained by transgenerational silencing of histone genes7. These studies, however, focused almost exclusively on autonomous mechanisms within the germline. It is unknown whether somatic signaling may contribute to maintenance of germline immortality.Germ cells are the only type of cells within an organism that deliver genetic and epigenetic materials to the offspring. Germ cells are therefore distinct from the somatic cells in their ability to maintain totipotency for production of an entire organism upon fertilization, and their immortality to allow reproduction for unlimited future generations. In C. elegans ENDU-2 belongs to a conserved but less studied family of proteins containing proposed poly-U specific endoribonuclease (XendoU) domains. While human EndoU  is used as a cancer marker gene, Viral EndoU Nsp15 is highly conserved in all known coronaviruses and has been suggested to promote viral RNA replication and limit innate immunity response of the host cells9. C. elegans ENDU-2 has been shown to regulate cold stress response10. Very recently, ENDU-2 was reported to regulate nucleotides metabolism and germline proliferation in response to alterations in nucleotide levels and genotoxic stresses11.Here we report that secretion of the poly-U specific endoribonuclease ENDU-2 from the soma to the germline preserves germline immortality at elevated temperature. We find that ENDU-2 binds to mature mRNAs and downregulates mRNA levels both in the soma and in the germline. In addition, RNA-binding and -cleavage are two separable activities utilized by ENDU-2 to control gene expression via distinct mechanisms. In the soma, ENDU-2 relies on its endoribonuclease activity to negatively regulate a subset of its mRNA targets. In the germline, ENDU-2 prevents misexpression of soma-specific genes and ensures stem cell immortality primarily via its RNA-binding activity, suggesting an essential role of ENDU-2 in retaining the stem cell-specific program of gene expression. In summary, our data suggest that the soma sends ENDU-2 as a messenger to the germline to control gene expression across tissue boundaries in response to temperature alterations.endu-2(lf) mutants were phenotypically similar to wild type animals in the first few generations, except displaying slightly reduced germline proliferation, an egg-laying defect (Egl) due to abnormal development of the vulva, and a moderate reduction in adult lifespan at 20 °C  mutants showed gradually increased sterility that, however, could be reset by additional outcrosses with wild type animals. We examined the fertility of two endu-2 alleles tm4977 and by188 over generations after four additional outcrosses. tm4977 allele has a 620 bp deletion starting from the 5′UTR to the end of the third intron, while the 20 bp deletion in by188 causes an early stop codon in the first exon. Therefore, these two alleles are probably null mutants. Both tm4977 and by188 alleles became sterile at 20 °C after about 15–20 generations  and increased number of apoptotic corpses  , whereas wild type animals had completely switched spermatogenesis to oogenesis. Furthermore, endomitosis occurred in the mitotic region   additionally showed high incidence of male progeny  and increased occurrence of other phenotypes . The latter were probably caused by elevated somatic mutation rate, since these phenotypes were not heritable. The sterile phenotype was 100% penetrant at 25 °C already at generation 6–10, but could not be detected at 15 °C  Fig. . We also25) Fig. . In the endu-2 in somatic tissues10, but did not report ENDU-2 localization in the germline. To retest the expression pattern of ENDU-2, we generated several independent transcriptional and translational endu-2 reporters with EGFP as well as a CRISPR/Cas9 EGFP knock-in strain at the endogenous endu-2 locus  resulted in strong localization of ssENDU-2::EGFP only in the intestine but not in the coelomocytes , muscles (myo-3 promoter) or intestine (vha-6 promoter), ENDU-2::EGFP was always detected in the coelomocytes  animals  wild type parents with either males or hermaphrodites of endu-2(−/−) that had been grown at 25 °C for five to seven generations and displayed strongly reduced brood size and high percentage of sterility  mothers had brood sizes with typically more than 100 F2 animals, the brood size of F1 cross-progeny derived from endu-2(−/−) mothers was as low as that of their mothers. We conclude that ENDU-2 function is required predominantly in the oocytes to preserve germline immortality.To know whether ENDU-2 affects oocytes or sperm to ensure germline immortality, we tested the parental contribution of ENDU-2 during sexual reproduction. For this purpose, we crossed ity Fig. . Whereasendu-2 mutants resembles that of hrde-1 mutants in the nuclear RNAi pathway15. In addition to loss of germline immortality, hrde-1 is also defective in multigenerational inheritance of germline RNAi. To test whether ENDU-2 acts in the nuclear RNAi pathway, we examined oma-1 RNAi inheritance that suppresses embryonic lethality of an oma-1 gain-of-function mutant for several generations16. Unlike the hrde-1 mutants that lost inheritance of oma-1 RNAi within two generations, effect of oma-1 RNAi knock-down persisted for 5–6 generations both in endu-2(lf) and wild type animals . Since wild type ENDU-2 would potentially cleave its RNA targets, we thought that this might prevent enrichment of intact RNA targets and their subsequent identification. Although we had no direct proof of an RNA cleaving activity of ENDU-2 yet, we reasoned that generation of an ENDU-2 variant that maintains RNA binding, but has lost RNA-cleavage activity, should facilitate RNA target detection. Xenopus XendoU mutants with E to Q exchanges at positions 161 or 167 of the EndoU domain lose RNA cleavage without reducing their RNA-binding activity17. The second glutamic acid (167E) is conserved in both XendoU domains of C. elegans ENDU-2 (175E and 460E), whereas the first (161E) is only found in the second XendoU domain (454E)  background and performed RIP-Seq with these two strains in additional to wild type ENDU-2::EGFP. By plotting normalized reads (RPM) of each transcript we were able to investigate RNA-binding affinity of ENDU-2 under different conditions. In general, all three ENDU-2 variants tested showed stronger RNA-binding affinity at 15 °C than at 25 °C  animals. In addition, expression of the wild type endu-2::EGFP transgene, but not of endu-2(E454Q)::EGFP, restored RNA decay in endu-2(lf) mutants, supporting our hypothesis that ENDU-2(E454Q) lost RNA-cleavage capacity despite its increased RNA-binding affinity. Surprisingly, extrachromosomal expression of ENDU-2(E454Q), but not of ENDU-2(E460Q), fully rescued the Mrt phenotype of endu-2(lf) at 25 °C  and endu-2(+) backgrounds suggested a similar number of transcripts being up- and downregulated by endu-2 (fold-change >2)  animals, germline expressed genes (n = 206) were over-represented  mutants at 25 °C became significantly smaller  Fig. . Among tendu-2(lf) background. Thirty-two percent (76 out of 237) of these transcripts were candidates for direct ENDU-2 targets, since they had been co-immunoprecipitated with ENDU-2 (Supplementary Data endu-2(lf) animals  phenotypes were temperature-dependent, we asked whether ENDU-2 regulates mRNA abundance in response to temperature alterations. To test this, we performed qPCR to quantitate ten selected mRNAs that were bound and downregulated by ENDU-2. We found that mRNA levels of these targets were upregulated in endu-2 mutant background, compared to wild type, already at standard growth temperature (20 °C), and were even more abundant at 25 °C  animals had higher fat-7 transcript levels than wild type at both 20 and 25 °C  displayed stronger FAT-7::GFP expression levels at both temperatures. Furthermore, transgenic expression of endu-2(wt) but not the endu-2(E454Q) transgene (that has lost RNA-cleavage activity) restored decreased FAT-7::GFP level  animals which had been grown at 25 °C from L1 stage, and sequenced two biological replicates of polyadenylated RNA from each strain. A gene with normalized reads of RPKM > 1 was scored as being expressed. We identified 8356 expressed genes in the gonad of wild type animals at 25 °C, among which 88% (7365) have been reported as expressed in the wild type gonad at 20 °C19. The following DESeq2 analysis showed that upregulation of mRNA levels was the prevalent change in the gonad of endu-2 mutants  background were enriched in ENDU-2 binding, as 218 out of 422 upregulated genes  and 11 out of 35 downregulated genes  were co-immunoprecipitated with ENDU-2. We considered them as direct targets of ENDU-2 and grouped them in two classes, depending on whether they are up- (class I) or downregulated (class II) in endu-2(lf) background  for each transcript to estimate the expression ratio between the soma and the gonad. Class I, but not class II, targets had on average significantly higher SEF values than the total mRNAs  mutant carrying the endu-2(E454Q)::EGFP transgene. Expressing endu-2(E454Q)::EGFP was sufficient to repress germline expression of most class I targets . We could demonstrate that the tissues containing ENDU-2 protein differ from those that harbor endu-2 mRNA and presence of a secretion signal peptide is necessary and sufficient to target ENDU-2 to the secretory pathway to reach distant cells.Our data implicate ENDU-2 in enabling long-range communication between cells, mediated via its secretion signal peptide Fig. . Whereaseak Fig. . We compOur data suggest that ENDU-2 production and function may occur in different tissues, and that secretion of ENDU-2 allows the control of mRNA abundance in the distance. A simple assumption would be that ENDU-2 has similar functions in any of its target tissues, no matter from which cells it is expressed. However, only neuronally or intestinally expressed ENDU-2 was able to rescue germline immortality Fig. , indicatendu-2(lf) mutants at elevated temperature  strain was used as wild type in all experiments in this study. Mutant strains: KHR83 endu-2(tm4977), outcrossed 8x with our N2. BR7130 endu-2(by188), NL3511 ppw-1(pk1425), TX20 oma-1(zu405), BR7510 oma-1(zu405);endu-2(tm4977), BR8649 oma-1(zu405);hrde-1(tm1200), BR7205 endu-2(by190[endu-2::EGFP]), BR7089 byEx1315, BR8657 endu-2(tm4977);byEx1814, BR7295 endu-2(tm4977);byEx1375, BR5402 byEx749, BR8672 byEx1821, BR7827 endu-2(tm4977);byEx1551, BR7332 endu-2(tm4977);byEx1379, BR8551 endu-2(tm4977);byEx1795, BR8662 endu-2(tm4977);byEx1816, BR7512 endu-2(tm4977);byEx1449, BR8747 endu-2(tm4977);byEx1847, BR8821 endu-2(tm4977);byEx1875, BR7680 endu-2(tm4977);byEx1492, BR7683 endu-2(tm4977);byEx1495, DMS303 nIs590[fat-7p::fat-7::GFP + lin15(+)], BR8317 endu-2(tm4977);nIs590[fat-7p::fat-7::GFP + lin-15(+)], BR8754 endu-2(tm4977);nIs590[fat-7p::fat-7::GFP + lin-15(+)];byEx1853, BR8775 endu-2(tm4977);nIs590[fat-7p::fat-7::GFP + lin-15(+)];byEx1860, RT688 unc-119(ed3);pwIs28[pie-1p::cav-1::GFP(7)+unc-119(+)], BR8358 endu-2(tm4977);pwIs28[pie-1p::cav-1::GFP(7)+unc-119(+)]. For RIP-Seq the following strains were used: BR7802 endu-2(tm4977);byIs240, BR7803 endu-2(tm4977);byIs241, BR7205 endu-2(by190[endu-2::EGFP]) and Ex[ife-2P::GFP] . For the RNA-cleavage assay, BR8311 endu-2(tm4977);byIs267 and BR7803 endu-2(tm4977);byIs241 were used in additional to N2 and endu-2(tm4977) strains. endu-2(tm4977) allele was used for all experiments, if not noted otherwise. Except for the Fig. endu-2(lf) mutants were the granddaughter generation (G2) from an endu-2(tm4977) carrying transgenic endu-2 rescue strains whose parents (G1) had lost the rescue transgene. Information for extrachromosomal transgenic strains generated in this study is summarized in the Supplementary Data The endu-2 (pBY3798), a 4691 bp genomic fragment upstream of the endu-2 ATG was PCR amplified and inserted into pEGFP-N1 with Eco47III/BglII sites. For translational fusion reporters, 5487 bp endu-2 genomic region was cloned into pBY3798 with BglII/SmaI sites to receive endu-2P::endu-2::EGFP (pBY3800), the endu-2 3′UTR was inserted into pBY3800 at NotI site to receive endu-2P::endu-2::EGFP::endu-23′UTR (pBY4137). Δssendu-2::EGFP (pBY3843) and Δssendu-2::EGFP::endu-23′UTR construct (pBY4172) was generated by removing sequences encoding the N-terminal 2–19 amino acids from pBY3800 and pBY4137, respectively. The endu-2P::SSsel-1::Δssendu-2::EGFP construct (pBY4194) was generated by inserting the sequence encoding the amino acids 1–20 of sel-1 into the BglII site of pBY3843. A endu-2P::3xFlag::endu-2::EGFP::endu-23′UTR expressing construct (pBY4138) was generated by inserting 3xFlag encoding sequence and endu-2 genomic region into pBY3798 with BglII and BglII/SmaI sites, respectively. The 3′UTR of endu-2 was inserted via Gibson ligation. The unc-119P::EGFP expressing construct (pBY2941) was made by fusing the 2.1 kb unc-119 promoter region into pEGFP-N1 with Eco47III/BglII sites. The unc-119P::SSendu-2::EGFP construct pBY4148 was generated by inserting the first 57 nucleotides of endu-2 cDNA into pBY2941 with BglII/AgeI sites. For expressing recombinant ENDU-2::EGFP in HEK 293T cells, wild type endu-2 cDNA was cloned into a 3xFlag containing pEGFP-N1 with XhoI/SmaI (pBY3878). Site-directed mutagenesis was performed to receive endu-2(E454Q)::EGFP::3xFlag (pBY3894) and endu-2(E460Q)::EGFP::3xFlag mutants (pBY3895). The fragments endu-2::EGFP::3xFlag from pBY3878, endu-2(E454Q)::EGFP::3xFlag from pBY3894 and endu-2(E460Q)::EGFP::3xFlag from pBY3895 were inserted into pBY3798 to receive constructs for generating endu-2P::endu-2::EGFP::3xFlag (pBY3892), endu-2P::endu-2(E454Q)::EGFP::3xFlag (pBY3897) and endu-2P::endu-2(E460Q)::EGFP::3xFlag (pBY3898) reporter strains. EGFP::3xFlag fragments in pBY3892 and pBY3897 were removed to receive pBY4188 endu-2P::endu-2 and pBY4066 endu-2P::endu-2(E454Q). Somatic gonad specific expression was achieved via PCR amplifying and insertion of endu-2 cDNA into a fos-1P::GFP plasmid (a gift from David Sherwood lab) with SalI and SmaI sites (pBY3833). For intestinal specific expression, a 1.2 kb vha-6 promoter was inserted at Eco47III/XhoI sites into pBY3878 to receive vha-6P::endu-2::EGFP::3xFlag (pBY3937). A 2.1 kb unc-119 promoter was inserted into pBY3878 at Eco47III/BglII sites to receive unc-119P::endu-2(cDNA)::EGFP::3xFlag (pBY4127) for neuronal expression. A 2.4 kb myo-3 promoter region was cloned into pBY3878 to receive myo-3P::endu-2::EGFP::3xFlag (pBY4135) for muscular specific endu-2 expression.To construct an EGFP transcriptional fusion reporter of endu-2(tm4977) and endu-2(by188) were further outcrossed 4× with N2 before scoring Mrt phenotype for Fig. endu-2(tm1977) animals were maintained at 25 °C for 6 generations until endu-2(tm4977) showed strong sterile phenotype. Fifteen L1-L2 animals from the 6th generation were shifted to 15 °C and designated as G1. For Mrt rescue experiment, endu-2(tm4977) progenies of endu-2(tm4977);byEx1375, endu-2(tm4977);byEx1449, endu-2(tm4977);byEx1875, endu-2(tm4977);byEx1488 and endu-2(tm4977);byEx1488 animals (P0) were isolated and designated as G1.Mrt phenotype was examined by quantification of brood size by separating 15 L4 animals on single plates and counting number of progeny per animal, or transferring six L4 larvae onto new agar plates and scoring percentage of the fertile animals 36 h after the L4 stage in the next generation. Both The ‘number of nuclei in the proliferative zone’ included all the germ nuclei between the distal tip and the transition zone of day-one adult animals . For visualizing and counting germ cell nuclei in the proliferating zone of the gonad, dissected gonads were fixed in methanol and suspended in PBST containing 0.1% Tween 20 and 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) before microscopy. Z-stack images of animals were collected and the numbers of the germ cells were counted with a publicly available ImageJ Cell Counter plug-in originally written by Kurt De Vos at the University of Sheffield, Academic Neurology.endu-2(tm4977) animals display strong egg-laying defect due to abnormal vulval development, agar plates containing 200 µM FUDR were used to during the first seven days of adulthood to avoid internal hatching.Lifespan at 20 °C was initiated at L4 stage. As 29. A 1:200 dilution of a monoclonal mouse anti-GFP antibody  and a 1:25 dilution of the secondary antibody were used to detect ENDU-2::EGFP in the gonad.Antibody staining of dissected gonads was performed with day-one adult animalsoma-1 RNAi was initiated at L1 stage at 20 °C (restrictive temperature) and these animals were designated as P0. Six P0 day-one adults animals were transferred onto OP50 seeded agar plates and incubated at 20 °C for scoring the embryonic lethality over generations. Embryonic lethality was examined by transferring of about 100 embryos onto an OP50 seeded agar plate and quantifying the number of hatched animals after 48 h.Worms were fixed in 500 µl 60% isopropanol. After removal of supernatant, 500 µl freshly filtered ORO working solution was added to stain worms at 25 °C in a wet chamber overnight. Animals were washed once with 1 ml 0.01% Triton-x100 containing M9 buffer and responded in 250 µl M9-Triton-x100 buffer. Stained worms can be stored at 4 °C for at least 1 month. For quantification, color images were recorded. The original images were spitted into red and blue channels and the level of Oil Red-O was quantified by determining the excess intensity in the red channel in comparison to the blue channel. The mean lipid content per worm was calculated as the total intensity within stained regions normalized by the area of the worm regions. Oil Red O working solution was prepared as follows: 0.5 g of Oil Red O powder was dissolved in 100 ml isopropanol solution and equilibrated for several days. The solution was then freshly diluted with 40% water for a 60% stock and allowed to sit overnight at room temperature and filtered using 0.2 mm filters.g). Lysates were clarified by centrifuging at 16,000 × g for 15 min. Supernatants were pre-cleared with Dynal Protein A magnetic beads (Invitrogen) and incubated with anti-GFP antibody (Abcam) for 1 h at 4 °C, followed by incubation with Dynal Protein A Magnetic Beads for 2 h at 4 °C. Beads were washed three times with Lysis-NP-40 Puffer before DNase I digestion  at 37 °C for 30 min. In all, 10 µl of 50 mM EDTA was added to stop DNase digestion. RNA was extracted with 5 volumes Trizol reagent (Invitrogen), followed by isopropanol precipitation. Precipitates were resolved in RNase-free water for visualization on 12.5% denaturing acrylamide gel with SYBR Gold (Thermo Scientific). Random hexamer primers were used for preparing TruSeq3 reverse-stranded cDNA libraries for Illumina single-end 50 bp sequencing. RIP-Seq to compare RNA-binding efficiency of ENDU-2(wt), ENDU-2(E454Q), and ENDU-2(E460Q) at 15 and 25 °C were performed without cross-linking. RIP-Seq to determine RNA targets of ENDU-2 was carried out with ENDU-2(E454Q)::EGFP expressing animals raised at 15 °C with UV cross-linking.To isolate ENDU-2 associated RNAs, animals of mixed stage were lysed in Lysis-NP-40 buffer  with SilentCrusher S and the WS260 canonical gene set  with the RNA aligner STAR 2.6.0b32 with default settings, then counted reads mapping to individual genes using feature Counts 1.6.233. For a read to be counted as mapping to a particular gene, we required a minimum read mapping quality of 12 (-Q 12 option of feature Counts) and an overlap of at least 1 base between the read and any of the exons of the gene (--minOverlap 1).The sequencing data were uploaded to the European Galaxy Server at 6. The addition of 1 in the FC formula prevents infinite enrichment. ENDU-2 associated RNAs were defined as transcripts with both FC > = 4 and RPKM > = 1 in ENDU-2(E454Q) IP sample. RPKM is calculated as follows: RPKM = RPM/length of transcripts × 103.The IP enrichment of a transcript was expressed as FC = (IP RPM + 1)/(Mock IP RPM + 1), where RPM = number of reads mapped to a gene/ × 10https://usegalaxy.eu. All read quality controls, trimming, mapping and counting were performed through Galaxy30 using protocols like described for RIP-Seq (above). The DESeq2  was used to determine differentially expressed features from count tables of differential transcript abundances. The RPKMs throughout this study were normalized separately. Only the longest isoform of each gene was used as length of a transcript. To estimate genes with enriched expression in the soma, RNA-seq data from wild type animals raised at 25 °C from a previous study20 were compared with our transcriptome from isolated gonads of wild type animals. The soma enrichment factor (SEF) was calculated with the formal SEF = /(RPKMgonad + 2)]. Addition of factor 2 to the formal minimize large SEF value caused by low RPKM values.Animals were raised at 25 °C from the L1 larval stage and the complete gonads were isolated 24 h after mid-L4 stage. Total RNA of the gonads was extracted with RNeasy® Mini Kit . Purification of poly-A containing RNA molecules, RNA fragmentation, strand-specific random primed cDNA library preparation, and single-read sequencing (50 bp) on an Illumina HiSeq 4000 were carried out by Eurofins Genomics. The RNA-seq results from two independent biological replicate of dissected gonads were uploaded to the European Galaxy Server at 34. fat-7 and trcs-1 mRNA were transcribed from complementary DNA (cDNA) with biotinylated uracil. The presence and purity of RNA were examined on an RNA gel. This biotin-labeled RNA was incubated with recombinant ENDU-2(wt)::EGFP::3xFlag, ENDU-2(E454Q)::EGFP::3xFlag, and ENDU-2(E460Q)::EGFP::3xFlag proteins expressed in HEK 293T cells. The labeled RNA was captured and isolated using streptavidin beads. Proteins binding the RNA were then visualized via western blot. The uncropped blots are included in the Supplementary Fig. The RNA-binding assay was performed as describedg). 5 µl RNAsin Plus RNAse Inhibitor (40 U/µl) was added to the worm lysate and centrifuged by 15,000 × g for 15 min at 4 °C. For the RNA-cleavage assay 95 µl supernatant of the worm lysate with 200 µg total protein was incubated with 5 mM Ca2+, Mg2+, Mn2+, or 10 mM EGTA for 30 min at 7 °C before total RNA was extracted with Trizol. 0.2 µg RNA was loaded on 10% 8 M Urea acrylamide gel with SYBR Gold for visualization of RNA.Pellets of animals raised at 15 °C in mixed stages were washed twice with 1 ml 10 mM EGTA containing M9 buffer before resuspension in 0.7 ml NP-40 buffer. 3x Protease inhibitor was added to the buffer and worms were lysed with SilentCrusher  and endu-2(tm4977);byEx1375 animals used for microarray were granddaughter decedents of one single endu-2(tm4977);byEx1375 animals 35.GO term and tissue enrichment analysis was carried out with the online enrichment analysis tool (endu-2(tm4977) animals were granddaughter decedents of endu-2(tm4977);byEx1375 animals. act-4 was used as internal control. qPCR primer sequences: cav-1 forward aagtgctggtggagtagatgc, cav-1 reverse tccgatagcgatgttctcttc, clec-169 forward tggacacttgtaactgtgcaaga, clec-169 reverse ttttcatttgaccactttagatcg, F38B6.4 forward cccttgtcctcggaaattaga, F38B6.4 reverse caggcccgaagatggtaat, pfas-1 forward caagattggaagttccgaaga, pfas-1 reverse ctcaatatccggacagtcgtc, B0286.3 forward gacccgaaaacttggagctt, B0286.3 reverse Tggaacgaatgagcacataatc, atic-1 forward tgctacaaaaatgccagcag, atic-1 reverse cgtttaaaggaagaccaacagc, acox-1.2 forward agcggtgatctatggaagtga, acox-1.2 reverse agctcagggatcttggacac, abdh-3.2 forward atgacaccaccccaattgtc, abdh-3.2 reverse tgctgtcatgagtacttcctgtg, col-176 forward ccacaacctttgctccaatc, col-176 reverse gagcacacttgatgcagtcg, clec-15 forward tgcgccagaagggtattact, clec-15 reverse cgcagaacgatctaaccaga, asp-14 forward gctgcagttaccaacattacca, asp-14 reverse agcaacgaagaaggttgagg.Total RNA was prepared from day-one adult animals raised at 25 or 20 °C by using RNeasy® Mini Kit . endu-2(tm4977) L4 animals were selected and dissected 24 h later for smFISH staining using a published protocol procedure36. Images were acquired with an Image Z1 fluorescence microscope. Exposure times and acquisition settings were identical between replicates. mRNA puncta were quantified by using ImageJ 1.51 s cell counter plug-in. Regions of interest for acquisition were defined by nuclei DAPI staining.In total, 38–48 probes of 20 nt in length targeting each mature mRNA were designed with Stellaris RNA FISH probe designer on gene target specificity. Probes were produced, conjugated to CAL Fluor Red 610, and purified by Biocat (sequences of the probe sets are available in Supplementary Data P-values, statistical significance, number of biological replicas, and sample sizes for all of the graphs are indicated in the figures or figure legends. n represents the number of animals tested, unless mentioned otherwise. N means the number of biological replicates. ***P-value <0.0001.Experiments shown in this study were performed independently 2–4 times. Details of the particular statistical analyses used, precise Further information on research design is available in the Supplementary informationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting SummaryPeer Review FileDescription of Additional Supplementary Files


Functional enrichment analysis of these lncRNAs revealed that they were involved in the regulation of many key signaling pathways during ovine oocyte development, which was reflected by their targeted genes. From this study, multiple lncRNA–mRNA networks were presumed to be involved in key signaling pathways regarding ovine oocyte maturation and meiotic resumption. In particular, one novel lncRNA (MSTRG.17927) appeared to mediate the regulation of phosphatidylinositol 3-kinase signaling (PI3K) signaling during ovine oocyte maturation. Therefore, this research offers novel insights into the molecular mechanisms underlying ovine oocyte meiotic maturation regulated by lncRNA–mRNA networks from a genome-wide perspective.Long non-coding RNA (lncRNA) is involved in many biological processes, and it has been closely investigated. However, research into the role of lncRNA in ovine ovarian development is scant and poorly understood, particularly in relation to the molecular mechanisms of ovine oocyte maturation. In the current study, RNA sequencing was performed with germinal vesicle (GV) and in vitro matured metaphase II (MII) stage oocytes, isolated from ewes. Through the use of bioinformatic analysis, abundant candidate lncRNAs in stage-specific ovine oocytes were identified, and their  In mammals, fully grown follicles release mature oocytes that are ready for fertilization. Each oocyte is enclosed in a follicle and undergoes an extended period of meiotic arrest that may last 40 years or more in humans and some other animals , during A long period of meiotic prophase or GV arrest is suggested to be of great importance for the production of a fertilization-competent oocyte . The maiNon-coding RNAs (ncRNAs) are generally divided into two classes according to length: short/small ncRNAs [such as micro-RNAs (miRNAs)] and long non-coding RNAs (lncRNAs) with a length generally exceeding 200 bp . Followiin vitro were sequenced for the expression of lncRNA. Through RNA data analysis, the stage-specific lncRNAs and coding genes were identified. Furthermore, the co-expression (trans) and co-location (cis) analyses were implemented with differentially expressed lncRNAs (DELs) and genes (DEGs) to explore the regulatory networks involved in oocyte transition from GV to MII stages.RNA transcriptome sequencing is now one of the most powerful methods available for the detection and expression analysis of lncRNAs . In the All the experiments in this research followed national guidelines and were in accordance with the Ethical Committee of Anhui Agricultural University. All surgery on ewes had the approval of the Institution of Animal Care and Use Committee, Anhui Agricultural University (no. 2016017), China.in vitro with maturation medium in a humidified incubator at 38.5°C with 5% CO2 for 23–25 h. MII stage oocytes with an extruded first polar body were harvested. The sequence samples were prepared with 30–35 oocytes as one biological replicate.Ovine ovaries were collected from a slaughterhouse in Ürümqi in Xinjiang, China. They were then transported in saline solution supplemented with penicillin–streptomycin to the laboratory within 4 h. There, follicles of 2–8 mm in diameter were extracted from the washed ovaries. After settlement for a short time, the supernatant was removed, and the cell pellet was resuspended with washing buffer for subsequent dissection and selection under a stereoscopic microscope. The oocytes were divided into two classes: (i) denuded oocytes (GV stage) and (ii) oocytes surrounded by cumulus cells; the latter were treated with hyaluronidase  to remove the cumulus cells. Oocytes were then cultured 3, 0.2384 g HEPES, 0.022 g Na-Py, 0.0075 g penicillin, 0.005 g streptomycin, and 0.003 g glutamine supplemented with 100 IU FSH, 1 ml 17β-E2 (working concentration is 1 μg/ml), and 200 IU LH. Phenol red was added to adjust the pH value, and the medium was filtered through a 0.22 μm filter and preserved at 4°C.As in previous reports , the mat® spectrophotometry  for detecting RNA purity, Qubit® 2.0 Fluorometer  for quantifying RNA concentration, and RNA integrity assessment with an RNA Nano 6000 Assay Kit and a Bioanalyzer 2100 system . An aliquot of 3 μg RNA from each sample was used as the input to prepare the sequence. After removal of ribosomal RNA, the sequence libraries were generated with a NEBNext® UltraTM Directional RNA Library Prep Kit  according to the manufacturer’s recommendations, the quality of which was assessed with an Agilent Bioanalyzer 2100 system. After sample clustering with a TruSeq PE Cluster Kit v3-cBot-HS  according to the manufacturer’s instructions; sequencing was conducted on an Illumina HiSeq 2000 platform using a 100 bp paired-end strategy.RNA samples from the collected oocytes were extracted with TRIzol reagent, and the following pipeline was performed to ensure RNA quantity: 1% agarose gel electrophoresis for RNA degradation and contamination test, NanoPhotometer1, clean data were aligned to the sheep reference genome (version Oar_v3.1 from ensemble) to produce Bam files. After Bam files were sorted with SAMtools (v1.9)  . FastQC s (v1.9) , transcrs (v1.9) . Subsequ2 (v0.11.2). The produced transcript file may then contain the lncRNA transcripts with protein coding potential that should be removed. Here, we predicted the protein coding ability of lncRNA transcripts through three methods: Coding–Non-coding Index (CNCI)  , Coding x (CNCI) , and Pfax (CNCI) . The comp-values of the transcripts were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). Significant DEGs and DELs were determined with padj  <0.05.The expression levels of mRNA and lncRNA were calculated using featureCounts (v1.6.4)  based ontrans-role through their related co-expression genes (r) using the R package Hmisc (v4.3)3. Those mRNAs with a p-value < 0.01 and |r-value| > 0.9 were considered as co-expressed genes of their lncRNAs. Further, for cis-regulation, based on the assembled GTF file of candidate lncRNAs, we employed FEELnc (v 0.1.1)   to scree4 (5 (Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed predominantly based on Metascape4 , a web-b4  for enri4 . Given t4  and COCs were isolated from ovine ovaries. Cumulus cells were removed from COCs and the isolated oocytes developed to the mature MII phase during a 23–25 h incubation quencing . Represequencing , the clequencing . In partquencing , which wAccording to data processing, more transcripts of coding genes were generated than lncRNAs . To undep-value  and Pearson’s correlation coefficient , 173.268 pairwise lncRNA–mRNAs were produced  were common with co-expressed lncRNAs  might be involved in the functional regulation of calcium ion transport and cAMP response. It has been well demonstrated that calcium in many species is involved in oocyte meiotic maturation . Furthermore, several cis-regulatory pathways of key lncRNAs were identified, which included insulin secretion, Wnt, Hippo, cGMP-PKG, and calcium signaling  can be found below: The animal study was reviewed and approved by the Institution of Animal Care and Use Committee, Anhui Agricultural University, China.J-JW, M-HN, and TZ conducted the experiments. J-JW analyzed the data. J-JW, H-GC, and WS wrote the manuscript. H-GC and WS designed the experiments. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution. Non‐enzymatic RNA Copying: Enhanced non‐enzymatic ligation allows the rapid copying of long RNA template and short RNA splinted templates, thus suggesting a potential route to the assembly of artificial systems capable of evolution. The RNA‐world hypothesis posits an early stage in the evolution of life during which RNA was both the carrier of genetic information and the catalyst of cellular reactions.As an alternative to mononucleotide polymerization, templates might be copied by the ligation of short oligomers to generate longer products.N2‐type reaction. Studies of both monomer polymerization and oligonucleotide ligation will help us to better understand how a hereditary material could replicate prior to the advent of ribozymes.Herein, we explore the use of highly reactive oligonucleotide substrates in non‐enzymatic template directed ligation. We used short oligoribonucleotides terminated with 3′‐amino‐2′,3′‐dideoxy‐ribonucleotides as substrates because the 3′‐amino moiety is a stronger nucleophile than the 3′‐hydroxyl of ribonucleotides. Although there is no evidence that 3′‐amino‐2′,3′‐dideoxy‐ribonucleotides were prebiotically available, they are an excellent model for the study of how RNA oligonucleotides might behave if the nucleophilicity of the 3′‐hydroxyl could be enhanced, for example by assistance from small molecules or peptides capable of delivering a catalytic metal ion to the reaction center. 3′‐Amino nucleotides and oligonucleotides have been previously employed for origin of life studies.kobs max) of the RNA ligation reaction in the presence of 100 mm Mg2+ was 0.027 h−1 imidazole (HEI) is known to enhance the rate of non‐enzymatic polymerization of imidazole activated monomers.2+ on RNA ligation, we examined ligation reactions in the presence of different concentrations of Mg2+. To minimize the effect of Mg2+ on ligator binding, we performed reactions at saturating ligator concentrations, for example, 100 μm tetramer ligator or 10 μm decamer ligator. Mg2+ is thought to assist RNA ligation by deprotonating the 3′‐hydroxyl nucleophile.2+ was too slow to measure, while the ligation rate continued to increase up to approximately 0.1 h−1 at 1.6 m Mg2+ for the tetramer ligator, and around 0.3 h−1 at 1.2 m Mg2+ for the decamer ligator . Thus, we conclude that Mg2+ is beneficial for N3′‐P5′ ligation only when the concentration of the ligator is too low to afford sufficient binding to the template. Otherwise, the presence of Mg2+ decreases the rate of the reaction. Notably, the ligation rates of the decamer ligator are higher than the rates of the tetramer ligator. This effect may reflect a more stable and favorable conformation of decamer/template duplex.We also examined the effect of varying Mgm of the tetramer 2‐MeImp‐CUGA‐3′‐NH2. With HEI, we observed an approximately 20 % yield of full‐length product within 10 minutes and over 60 % by 40 minutes. Consistent with our previous results, at this saturating concentration of tetramer, Mg2+ inhibited ligation, leading to increased levels of a ladder of stalled products corresponding to the stepwise addition of tetramers. After two hours, we did not observe any further increase in the yield of full‐length product, and the stalled products remained unchanged in intensity. To test the hypothesis that the stalled products arise from templates occupied by hydrolyzed (i.e. unactivated) downstream ligator, we repeated the ligation experiments but added multiple aliquots of the coupling reagent EDC after the initial stage of the reaction was largely complete. In the absence of Mg2+, this strongly decreased the stalled products and significantly increased the yield of full‐length product . The effect of EDC was much smaller in the presence of Mg2+. We suggest that more prebiotically plausible chemistry for the re‐activation of ligator oligonucleotides that have become deactivated due to hydrolysis, such as the recently described isonitrile activation pathway,Encouraged by the greatly enhanced ligation rate resulting from the combination of a 3′‐amino nucleophile and the HEI organocatalyst, we applied these advances to the copying of RNA templates by multistep ligation. We first tested the copying of a 32 nt RNA template containing eight 5′‐UCAG‐3′ repeats  should be addressed to the authors.SupplementaryClick here for additional data file.


Taken together with a recent proposal that proteins in general tend to interact with RNAs similar to their cognate mRNAs, this suggests a mechanism for RAP binding. Using a reporter construct, we show that ACRO1 potently inhibits Pol II elongation in cis. We propose a novel mode of transcriptional regulation in humans, in which the nascent RNA binds Pol II to silence its own expression.Transcription elongation is a highly regulated process affected by many proteins, RNAs and the underlying DNA. Here we show that the nascent RNA can interfere with transcription in human cells, extending our previous findings from bacteria and yeast. We identified a variety of Pol II-binding aptamers (RAPs), prominent in repeat elements such as ACRO1 satellites, LINE1 retrotransposons and CA simple repeats, and also in several protein-coding genes. ACRO1 repeat, when translated  Transcription, as the first step, is tightly regulated, and RNA polymerase II (Pol II) progression along the gene is not smooth. Pol II pauses in the promoter-proximal region and also during elongation . The dynfunction ), or by function . To datekaryotes ,8: mousekaryotes  and downer cleft . Certainer cleft .cis-acting nascent RNA-borne signals on transcription. Bacterial riboswitches, located in the 5ʹ untranslated regions of mRNAs, can dynamically refold in response to ligand binding or temperature shift and promote transcription elongation or termination [A less-explored field is the impact of mination ,15. Simimination . Furthermination . Alternamination .Escherichia coli and Saccharomyces cerevisiae [Our laboratory has shown that nascent RNA can regulate transcription by direct binding to the transcribing polymerase in revisiae ,20. Shorrevisiae .In this work, we extend the findings from bacteria and yeast and show that human Pol II is amenable to regulation by RAPs in the nascent RNA as well. We performed a genomic SELEX experiment to look for RAPs encoded in the human genome. We focus on one of the most highly enriched SELEX sequences derived from ACRO1 satellites and show that ACRO1-derived RAPs are potent self-silencing elements.S. cerevisiae, since human Pol II could not be obtained in sufficient purity and quantity. Due to the high degree of conservation of the enzyme [S. cerevisiae Pol II core [We constructed an RNA library  represene enzyme  and the  II core , we assuin vitro  and can be co-immunoprecipitated with Pol II from HeLa lysates , bind human Pol II  lysates , vindicaMARK4 gene on chromosome 19 (E. coli and S. cerevisiae [A/TC/A)n simple repeats and the ACRO1 family of satellites.Although the selection procedure resulted in the successful isolation of RNAs binding to Pol II, no significantly enriched sequence was observed in the small sample of 200 clones, suggesting that the pool from the 7th cycle contained many diverse sequences. Therefore, we subjected this enriched pool to deep sequencing and computational analysis . Enricheosome 19 . The majosome 19 . The enrbona fide transcripts, but rather domains within longer RNAs with Pol II-binding potential. We were unable to detect stable transcripts derived from ACRO1 satellites in HeLa cells. Nevertheless, ACRO1 satellites have been reported to be expressed at very low levels in several epithelial cancers [The ACRO1 consensus repeat unit is 147 bp long and occurs as 1.3–2.4 kb and 256 bp long arrays within a 6 kb higher-order repeat structure containing portions of LINEs, LTRs and DNA transposons. We termed these higher-order repeats “ACREs” for ACRO1-containing repeat elements  and SI F cancers  and earl cancers .Figure in silico into protein and aligned globally with a fragment of Pol II CTD, 23 out of 49 amino acids were identical  and most convincingly also reflected the repetitive nature of the heptapeptide repeat (D of 600 nM [−23) was ascertained by comparing the RAP-derived amino-acid frequencies to those derived from random RNA sequences, all normalized to the respective number of codons in the genetic code (see Methods for details).We noticed that ACRO1 satellites are rich in codons for amino acids present in the Pol II subunit 1 CTD, especially proline, serine and threonine. When the ACRO1 consensus sequence was translated e repeat ). Furthef 600 nM . This isf 600 nM . Recentlf 600 nM . We theret al., it was not possible to narrow down the sequences responsible for disruption of transcription, though the effect was clearly dependent on the length of the LINE sequence. These results possibly indicate that LINEs contain sequences reducing their expression to avoid active invasion and damage of the genome caused by retrotransposition. The fact that RAPs were especially enriched in active full-length LINEs supports this hypothesis . We repeated the above-mentioned experiments and analyzed the role of RAPs in LINE silencing. As can be seen in Another class of repeats prominent in our selection were the LINE elements, which was especially interesting because they had previously been reported to disrupt their own expression . There aGFP-LacZ reporter system had no or only a minor effect on steady-state RNA levels . HoweveA levels  and SI FA levels  and SI FGFP-LacZ reporter resulted in similarly depleted GFP expression levels . In addin trans ). It is To distinguish between post- and co-transcriptional regulation, we monitored transcript levels upstream and downstream of the ACRO1 insertion by RT-qPCR ). We comMARK4 gene intron 13 . The rein vivo [Transcription is a central process in cellular life, and its regulation occurs at multiple levels. The number of proteins known to interfere with this process is large. Recently, we showed that RNA can also be a potent regulator of transcription, and that the nascent RNA contains signals that communicate with the transcription machinery in bacteria and yeast ,20. Genoin vivo . Using gin vivo , and a cin vivo . It shouRecently, we have demonstrated that nucleobase-density profiles of typical mRNA coding sequences match closely the nucleobase-affinity profiles of their cognate proteins, with anti-matching seen only in the case of adenine profiles . This fiFurthermore, it is possible that ACRO1 repeats are evolutionarily derived from the Pol II CTD mRNA to introduce an additional level of transcription regulation close to centromeres. ACRO1 elements are moderately abundant in the human genome and are mainly located in pericentromeric regions which are transcriptionally inactive. Their mobility could have been provided by the mobile elements contained within the ACREs . This would be a very recent acquisition as they can only be found in primates.i.e., adenine-rich codons tend not to bind cognate residues [If ACRO1 RNA binds to CTD because of a mutual relationship on the codon level, why does the CTD mRNA itself not appear among RAPs? As mentioned above, adenine is the only nucleotide with anti-matching density and affinity profiles, residues ,35,39. Tin cis, abrogating transcription. It has already elegantly been shown that the secondary structure of the nascent RNA affects the rate of Pol II transcription in vitro by inhibiting backtracking and thus preventing the polymerase to escape from pausing [bona fide transcripts but rather domains within potentially expressed RNAs that convey Pol II-binding capacity to their host transcripts. In the context of our experiments, RAPs are part of the nascent transcript interacting with Pol II in cis during transcription. We observed that their effect on transcription is additive and that the more RAPs are present on the nascent RNA the stronger the inhibitory effect .cis-acting sequences within the nascent transcript that affects transcription elongation. This might be an essential self-regulatory strategy for repeat elements to stay silent, enabling their survival in the genome during evolution. In addition, we hypothesize that RAP-mediated control of transcription might play a role in gene-regulatory processes, which depend on the rate of Pol II progression, such as alternative splicing and termination [Recently, circular intronic long noncoding RNAs were shown to accumulate at the site of transcription, associate with the elongating RNA polymerase and act as positive regulators of transcription . Here wemination . Indeed,in cis. We propose a novel mode of transcriptional control in human cells, wherein the nascent RNA binds to the transcribing Pol II making it elongation-incompetent . Similat Pol II . E. coliS. cerevisiae in an in vitro binding reaction as described in ref [st-5th cycles, RNA was added at 1 µM and protein at 100 nM. To increase stringency and competition, RNA was added at 1 µM and protein at 10 nM for the 6th and 7th cycles. The binding buffer contained 10 mM HEPES pH 7.25, 40 mM NH4SO4, 10 µM ZnCl2, 1 mM KCl, 10 mM DTT, 5% glycerol and 10 mM MgCl2.The genomic library was created as described previously , with hud in ref . For the2, 0.5% Nonidet P-40, 1 mM DTT, 100 U/ml RNAse inhibitor (Promega), 2 mM vanadyl ribonucleoside complexes solution, 25 µl/ml protease inhibitor cocktail for mammalian tissues) per 10 cm−1 and removed from the dish with a cell scraper. After 10 min on ice cells were centrifuged at 4°C, 1000 × g. Whole cell extracts were prepared for co-IP as described [HeLa cells grown in 10 cm dishes were harvested at 80% confluence with 1 ml lysis buffer . Pol II antibody recognizes the phosphorylated as well as the unphosphorylated form of Pol II the enzyme. The concentration of antibodies used for immunoprecipitations was 2 µl/ml2 of culture dish using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. After 24 h, fluorescence was monitored with AxioObserver Z1 microscope coupled to AxioCam MRm (Carl Zeiss MicroImaging) and RNA was extracted with TRI Reagent (Sigma).HeLa cells were grown to 70–90% confluence and transfected with 0.4 mg of plasmid per cm32P-labeled DNA probe was hybridized in ULTRAhyb-Oligo Buffer (Ambion) at 42°C overnight. The probe was 5ʹ-labeled with T4 PNK (NEB).Total RNA was separated on a 0.8% agarose gel containing 6.7% formaldehyde, capillary-blotted onto a Hybond-XL membrane  and UV-crosslinked. GFP-positive cells were quantified by FACSCalibur (BD Biosciences) and data were analyzed in Cyflogic  and SPSS (IBM) software. From each sample, fluorescence of 10,000 cells was measured and only GFP-positive events, as determined by mock-transfected cell fluorescence, were taken into account.150 pmol biotinylated Oligo(dT) (Promega) was bound for 10 min at room temperature to 0.6 ml MagneSphere® magnetic beads (Promega) prepared according to manufacturer’s instructions. 80 mg of total RNA was denatured at 65°C, 10 min, chilled on ice for 5 min and mixed with Oligo(dT)-beads solution. After 10 min incubation at room temperature, the beads were washed six times and Poly(A)+ RNA was eluted according to manufacturer’s instructions. Before washing of the beads, the first supernatant was taken as Poly(A)-RNA. Both fractions were ethanol-precipitated.Two milligrams of total RNA or 200 ng of Poly(A)-fractionated RNA was denatured with 200 pmol of random nonamers (Sigma) at 70°C for 10 min. The reaction was split in two, one without reverse transcriptase as a control. RT was performed at 45°C for 90 min using OmniScript (Qiagen). 1/40 of the total reaction was used for PCR and approximately 1/30 was used per qPCR well. qPCR was performed in Mastercycler® realplex (Eppendorf) with HOT FIREPol® qPCR Mix (Medibena) and primers specified in Supplementary Table S1. Transfection was controlled for by normalizing expression values to neo and subsequently all amplicons were normalized to GFP 1.ACRO1 translation was aligned globally against a fragment of human Pol II CTD of equivalent length via Needleman-Wunsch algorithm as implemented in Expasy lalign  using BL6 sets of random RNA sequences of equivalent lengths were generated. Each set was then translated using the universal genetic code and the Jensen–Shannon divergence (JSD) between the distribution of the obtained amino-acid frequencies and the distribution of amino-acid frequencies in human Pol II CTD was determined. The p-value was determined by comparing the distribution of JSD values in the case of random sequences against the RAP-Pol II CTD JSD. The RAP-Pol II CTD JSD was over 10 standard deviations lower than the average random-sequence JSD, yielding an estimated p-value < 10−23. As different amino acids are encoded by a different number of codons, the above analysis was performed by first normalizing the amino-acid frequencies in translated RAPs and random RNA sequences or Pol II CTD by the respective number of codons in the universal genetic code.The statistical significance of the enrichment of Pol II CTD amino acids in translated RAPs was evaluated by an analysis of random RNA sequences generated computationally using background frequencies of the four RNA nucleotides in the entire human genome. For the complete set of RAPs identified in this study, 10The ACRO1 sequence used in the reporter assay has been deposited in the Genbank with the number GenBank KF726396. The raw data are available for download on the Sequence Read Archive under BioProject accession PRJNA616423.Click here for additional data file.