The discovery of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated Cas endonucleases in bacterial and archaeal species allowed scientists to modify, utilized, and revolutionize this tool for genetic alterations in any species. Especially the type II CRISPR-Cas9 system has been extensively studied and utilized for precise and efficient DNA manipulation in plant and mammalian systems over the past few decades. Further, the discovery of the type V CRISPR-Cas12 (Cpf1) system provides more flexibility and precision in DNA manipulation in prokaryotes, plants, and animals. However, much effort has been made to employ and utilize the above CRISPR tools for RNA manipulation but the ability of Cas9 and Cas12 to cut DNA involves the nuisance of off-target effects on genes and thus may not be employed in all RNA-targeting applications. Therefore, the search for new and diverse Cas effectors which can precisely detect and manipulate the targeted RNA begins and this led to the discovery of a novel RNA targeting class 2, type VI CRISPR-Cas13 system. The CRISPR-Cas13 system consists of single RNA-guided Cas13 effector nucleases that solely target single-stranded RNA (ssRNA) in a programmable way without altering the DNA. The Cas13 effectors family comprises four subtypes (a-d) and each subtype has distinctive primary sequence divergence except the two consensuses Higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) that includes RNase motifs i.e. R-X4-6-H. These two HEPN domains are solely responsible for executing targetable RNA cleavage activity with high efficiency. Further, recent studies have shown that Cas13d exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. In addition to that, Cas13d has shown additional advantages over other Cas13 variants, structurally as well as functionally which makes it a prominent and superlative tool for RNA engineering and editing. Therefore considering the advantages of Cas13d over previously characterized Cas13 subtypes, in this review, we encompass the structural and mechanistic properties of type VI CRISPR-Cas13d systems, an overview of the current reported various applications of Cas13d, and the prospects to improve Cas13d based tools for diagnostic and therapeutic purposes. E. coli back in the year 1987 by the Japanese scientist, Yoshizumi Ishino but due to insufficient DNA sequence data, they were unable to elucidate the function of these repeated arrays  . Once it got transcribed, it interacts with the crRNA and forms a duplex structure that is found to play a critical role in guiding Cas9 to its targeted DNA  . Later, s (SRSR) . Soon afs (SRSR) . Anothereted DNA  and thiseted DNA . In 2012eted DNA . The maieted DNA . In 2014eted DNA . In the eted DNA . These seted DNA . Furthereted DNA . In 2016eted DNA . Finallyeted DNA . Both ofadaptation and spacer acquisition, where the small size of the invader genome is integrated into the CRISPR array proximal to the 5′ leader sequence; 2) biogenesis is the process in which CRISPR array generates pre-CRISPR RNA (pre-crRNA) which is then converted into mature crRNA and thus provides targeting specificity; and 3) interference, where the crRNA binds with the effector Cas protein and forms ribonucleocomplex which finds and binds the target invader’s genome with the help of crRNA (guide RNA) and finally causes cleavage or degradation of the invader’s genome (DNA/RNA)  DNA/RNA) .The new classification is based on the phylogeny arrangement and sequence composition of the effector modules and the distinctive Cas genes which are signature endonucleases for specific types and subtypes of the CRISPR-Cas system . These sClass 2 CRISPR systems are well known and most extensively studied class because of the simpler organization of effector modules, as it includes single-Cas effector protein. This single-Cas effector protein is a multi-domain and multi-functional protein that can be customized and fine-tuned to edit/modify any targeted DNA/RNA substrates with great specificity . Hence mStreptococcus pyogenes) spCas9] is the most useful system for targeted genome editing and is well-characterized, structurally, and functionally  molecule, and the protospacer-adjacent motif (PAM) sequence present at the 3′ end of the targeted DNA sequence  complex. This active RNP binary complex stochastically scans the PAM sequence (5′-NGG-3′) on the target DNA sequence and binds to it and forms an active ternary complex, this escorts the activation of two nuclease cleavage domain of the Cas9: the HNH domain and the RuvC-like domain. The HNH domain cleaves the complementary strand and the RuvC domain cleaves the non-complementary strand of the targeted DNA, to the gRNA sequence and thus generating Double-Stranded Break (DSB). The DSB then undergoes repair with two kinds of endogenous DNA repair mechanisms: non-homologous end-joining (NHEJ) pathway or the homology-directed repair (HDR) pathway  Makarov. NaturalThe type II CRISPR-Cas9 system is most widely used and most accepted because it is highly flexible to target and cleave any DNA sequence of the broad spectrum of species, to get an enviable editing outcome with unprecedented ease and specificity . DespiteFrancisella novicida which has a single signature effector protein, Cpf1 now known as Cas12a, and then it was first characterized by the group of a researcher in Zhang’s Lab at MIT in 2015. Further, researchers have found high diversity in the evolution of Type V system using in silico technique and further sub-classified the Type V system into 10 sub-types from Type V-A to Type V-I and the new series of novel Type V-U  system. Each subtype was found to have dissimilar Cas12 proteins because of distinct domain organization and diverge RuvC sequences, distinct targetable substrates, cleavage pattern, distinctive PAM sequence, and composition of gRNA  with the help of the secondary structure and form a binary complex. After the formation of binary complex, the PI domain of Cas12a recognizes the T-rich PAM (TTTN/TTN/YTN) sequence and start unwinding of the targeted dsDNA, which immediately initiate Watson–Crick base pairing between the “seed” sequence of the gRNA and the targeted DNA at the proximal end of the PAM sequence, resulting in the formation of R-Loop structure. The formation of the R-Loop structure causes conformational changes in the NUC lobe and this induces target dsDNA cleavage activity of RuvC domains. However, researchers have found that only one RuvC domain carries out cleavage activity of the target and non-target DNA strands (TS and NTS), not by both RuvC. The RuvC domain incises the NTS and TS sequentially at PAM distant sites and generates staggered 5, 7, or 10 nt 5′ overhang DSB. This DSB undergoes either the NHEJ repair pathway or the HDR pathway  Gao et .in vitro and in vivo studies before they can be employed for any bio-medical or therapeutic application. Not only that, to date, only Cas12a, Cas12b, and Cas12e crystal structures have been resolved but the other type V family Cas proteins have not been resolved  Like cas9, Cas12a protein is a bi-lobed protein that consists of recognition (REC) and nuclease (NUC) lobes. However, the NUC lobe of Cas12a was found to have two RuvC nuclease domains instead of HNH domain, and these RuvC domains are arranged in such a way that they can be superimposed; ii) The Cas12a is unique as it also has RNase activity and thus can solely dapper the pre-crRNA to mature-crRNA; iii) The Cas12a recognizes T-rich PAM sequences such as 5′-TTN-3′/5′-TTTN-3′/5′-YTN-3′ in the target DNA sites; iv) The Cas12a cuts at PAM distant sites i.e., away from the recognition site and generate staggered ends. These unique properties and advantages of Cas12a make the CRISPR-Cas12 system, simpler, more precise, and another valuable asset in the genetic manipulation toolbox. Further, as discussed earlier that each type of V-subtypes has dissimilar Cas12 proteins which showed distinct targetable substrates i.e., it can target double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and even single-stranded RNA (ssRNA). Because of multi-substrate specificity CRISPR-Cas12 system has been used in a wide range of applications such as genome editing, transcriptional regulation, DNA and RNA base editing, detection of nucleic acid (DNA and RNA), detection of small molecules, and the list is growing . Howeverresolved . Therefoin silico approach scientists discovered the Type VI CRISPR Cas system which has brought a new role to CRISPR to edit and target even RNA with great specificity. Like type II and type V CRISPR systems, type VI CRISPR systems use single signature Cas proteins called C2c2 effectors now known as Cas13a (trans-activating CRISPR RNA (tracrRNA) unlike Cas9 and Cas12. This Cas13-crRNA complex remains nucleolyticly inactive until it binds to the target ssRNA. Once the Cas13-crRNA complex binds to the targeted ssRNA it forms active ternary RNP complex. This ternary RNP complex undergoes conformational changes which trigger the two HEPN (HEPN1 and HEPN2) domains to come close proximity to each other and generate a single catalytic site where it subsequently cleaves targeted ssRNA bearing a complementary sequence following the protospacer-flanking site (PFS) (cis cleavage) but also has the ability to cleave another non-specific ssRNA (trans cleavage) regardless of the perfect base-pairing of the crRNA or the presence/absence of a PFS. This non-specific or trans cleavage activity is termed as “collateral activity” or “collateral damage” or “collateral cleavage”. On the other hand, such “collateral cleavage” by Cas13 has not been detected in mammalian cells and even in plants, making it a pro and robust tool for spatiotemporal transcriptional editing and manipulation in the eukaryotic system  . Like Cac system .The type VI Cas13 system is further sub-classified into four subtypes VI-A, VI-B, VI-C, and VI-D, based on the presence of adaptation genes (Cas1/Cas2), extra ORF region, associated accessory proteins, and finally due to the significant divergence at the primary sequence except for the conserved two HEPN domains sequence. Each subtype consists of respective Cas13 effectors designated as Cas13a (C2c2), Cas13b, Cas13c, and Cas13d. Moreover, Cas13a, Cas13b, and most recently discovered Cas13d have been well characterized  and have been used extensively in various RNA engineering applications. On the other hand, due to the lack of structural and functional information of Cas13c, making it a less comprehensive tool for RNA manipulation as compared to other Cas13 subtypes . HoweverEubacterium and Ruminococcus. Recently, the computational and cryoEM studies of Cas13d reveal that it is the smallest endoRNases with ≈930 amino acids in size and bears no significant sequence similarity to other Cas13 effectors except the two conserved R-X4-6-H HEPN motif (HEPN1 and HEPN2) domains  is now ready to actively search and identify the complementary targeted ssRNA  presence of any mismatches in the two separate crRNA spacer region  and 3′-end region (spacer nucleotides 13–22) with target ssRNA, founds to be intolerant and completely eradicate the ssRNA cleavage activity; iii) presence of Mg2+ and additional accessory component WYL domain increases the endoRNase activity; iv) Cas13d tends to cleave only structurally accessible ssRNA sequences, any secondary structure present in the target RNA sequence it fails to recognize and the RNA cleavage activity is abolished; v) Cas13d has exhibited no preference for PFS imposed ssRNA cleavage but has shown considerable preference for uracil bases in target ssRNA structures. Moreover, the vigorous cleavage activity of Cas13d has been found to remain functional through a broad range of temperatures i.e., 24–41°C and thus making it a potential tool for use in transcriptome engineering in a wide range of hosts  region, and 20 nucleotides, 3′ spacer region that is complementary to the target ssRNA. Upon generation of mature crRNA, Cas13d recognizes the 5′ crRNA handle/DR and clamped the first two base pairs (bp) of it with the help of NTD and HEPN2 domain while the 3′ spacer region remain sandwiched between the Helical-1 and Helical-2 domain. The HEPN1 domain act as a hinge by providing a structural scaffold to interconnect the 2 lobes of the Cas13d resulting in the formation of positively charged solvent-exposed RNA density. The binding of crRNA and Cas13d shows extensive molecular interaction between the sugar-phosphate backbone and nucleobases of Cas13d and the crRNA repeat region. These molecular interactions mainly occur in the 3′- end of the crRNA repeat region and are found to be crucial for correct positioning and proper binding of the crRNA with the Cas13d . Furthered ssRNA . The thrn plants   whereas ssRNA cleavage preferences for Cas13a are U- and Adenine (A) while ssRNA cleavage preferences for Cas13b are meant to be pyrimidine bases.Recently, the Cryo-EM structure of apo, binary, and ternary complex of Cas13a, Cas13b, and Cas13d has exposed several structural and functional distinctive features among them, which are discussed below, summarized in The newly discovered Cas13d effector has shown several advantages over other Cas13 variants in terms of structure as well as in function: i) Its small size which makes Cas13d an ideal and compatible for packaging into a viral vector and carried-out efficient delivery in the mammalian system; ii) Neurotoxicity effect of Cas13d founds to be less or absent in mammalian system; iii) Cas13d has dual nuclease activity i.e., it can cleave targeted ssRNA and can process maturation of crRNA simultaneously without any requirement of additional helper ribonucleases and tracrRNA; iv) Cas13d does not require protospacer flanking sequence (PFS) for RNA cleavage activity and this makes it lesser sequence constraints and can be used efficiently in targeting of “strongly depleted arrays”; v) Cas13d does not show off-target transcriptional perturbations as compared to other Cas13 variants and exhibits higher efficiency and specificity in cleaving targeted RNA in the mammalian system compared to other Cas13 endonucleases of the Cas13 enzyme family. These superlative features of Cas13d make it a prominent candidate for spatiotemporal transcriptome engineering, nucleic acid detection, multiplex gene regulation, post-transcriptional gene silencing, alternative splicing, tracking, and RNA epigenetic regulation .Soon after the discovery of type VI-D, scientists put extensive effort to characterize its biochemical, functional, and structural features because Cas13d (CasRx) is a small novel endoRNase that is highly specific, highly efficient, has autonomous pre-crRNA processing ability, and can be programmed to target any ssRNA sequence with no PFS requirement. This superlative quality of Cas13d makes it a prominent tool for transcriptome engineering, molecular diagnostic, and therapeutic development .Drosophila melanogaster and develop a programmable platform for RNA targeting of known phenotypic genes in addition to unexpected toxicity and lethality. They genetically encoded CasRx in flies and they found a moderate decrease in the targeted transcript. They also reported the cellular toxicity, off-target and on-target effects on the transcript cleavage by CasRx and they found that it cannot able to produce homozygous strains due to tissue necrosis and unexpected lethality arrived because of collateral cleavage of CasRx. Overall they have shown the limitation of using genetically encoded programmable RNA-targeting Cas13d system in Drosophila melanogaster and thus needs more extensive research and future optimization  efficiently and precisely degrade the specific mRNA transcript of a maternal and zygotic gene in zebrafish embryos. The efficiency of knock-down was observed to be 76% without producing any cytotoxicity and any abnormalities in development. Further, they also noted that there were no collateral cleavage activity and no direct off-target effects. Therefore, their study demonstrated that CRISPR-RfxCas13d can be utilized as an efficient knockdown tool to investigate the maternal and zygotic gene function in various animal embryos . Similarmization .in vivo and targeted the single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1). They successfully knockdown the Ptbp1, which resulted in the conversion of Muller glia into retinal ganglion cells (RGCs) and thus reducing the disease symptoms associated with RGC loss. They also reported a reduction in a motor defect in a Parkinson’s disease mouse model by inducing neurons with dopaminergic features in the striatum, using a CasRx-mediated approach with high efficiency. Therefore, conversion of glia to neuron using CasRx -mediated knockdown of Ptbp1 shows a potential therapeutic approach for tackling neurodegenerative diseases and brain damage due to neuronal loss  cell engineering of Chinese hamster ovary (CHO) cells. Their study demonstrated that Cas13d nuclease efficiently knock-down various exogenous and endogenous genes which are involved in apoptosis, gene amplification, metabolism, and glycosylation such as GS, BAK, BAX, PDK1, and FUT8 in CHO cells. They design specific gRNA for GS, BAK, BAX, PDK1, and FUT8 and co-transfected with Cas13d in CHO cells and they observed that the efficiency of knock-down of these genes was about 80–90% and was able to generate stable engineered CHO cells. Further, knockdown of the FUT8 gene causes attenuation of IgG fucosylation with 90% efficiency. Thus, their study has shown the generation of stable engineered CHO cells using the CRISPR-Cas13d platform, which has the ability to attenuate lactate accumulation and glycosylation and also shown apoptosis resistance which resulted in an increase in cell titer, enhance recombinant protein and antibody production during fed-batch culture . Similarin vitro. Further, they noticed that miR-497 and miR-195 jointly suppress the Hippo/Yap pathway by reducing the interaction between Yap and Smad3 and finally affecting the binding of the E2F4 which plays a critical role in silencing MIR497HG transcription in BCa cells. Their studies showed a potential strategy for the therapeutic treatment of BCa by knocking down of lnc-miRHGs MIR497HG using the CRISPR/Cas13d system  to target green fluorescent protein (GFP) RNA. They further optimize the approach by optimizing the inducer usage and manipulating guide RNA (gRNA) structural design to achieve the highest knockdown efficiency. However, they found that the highest knockdown efficiency of GFP at the mRNA level was ∼70% and at the protein level was 30–50% and considered it to be moderate repression. Moreover, this moderate repression of RNA is not only achieved by targeted RNA cleavage but also by the collateral cleavage activity toward bystander RNAs and thus torment bacterial metabolism (Due to the high activity of Cas13d (CasRx), Zhang et al., have recently employed the usage of the CRISPR-Cas13d system to carry out the knockdown of RNA in bacterial cells. They constructed an inducible expression system for CasRx with a tightly controlled promoter and a weakened ribosome binding site and incorporated it into tabolism .in vitro. Recently, Han et al., utilizes specific crRNA along with dead-RfxCas13d (dRfxCas13d), fused with modified plant peroxidase APEX2 (ascorbate peroxidase) with the help of the dsRNA-binding domain to target selected RNA, resulting in biotinylated labeling of transitorily interacting proteins and these interacting partners were analyzed using liquid chromatography-coupled tandem-mass spectrometry  which can recognize and bind to the target ssRNA but lacks cleavage activity. This dcas13d has further allowed the CRISPR researchers to utilize the CRISPR-Cas13d system beyond the RNA editing. Recently, Cas13d has been employed for the mapping of RNA–protein interactions trometry . Similartrometry .1-methyladenosine (m1A) and N6-methyladenosine (m6A) which are founds to regulate several cellular processes such as gene expression, alternate splicing, translation, and degradation of transcripts. Recently, Xie et al., developed a new CRISPR-Cas13d-based tool called “REMOVER”  to target specific transcripts for m1A demethylation. They introduced catalytically inactive RfxCas13d (dCasRx) and fused it with the m1A demethylase ALKBH3, and they found that ALKBH3 fused dCasRx protein can mediate specific demethylation of m1A of MALAT1 and PRUNE1 RNAs and also noted that it considerably increased their stability. Thus, their studies have established that REMOVER can be used as a tool for targeted RNA demethylation of specific m1A-modified transcripts. . In this method, they used catalytically inactive RfxCas13d (dRfxCas13d) fused with fluorescent-labeled crRNA along with the dCas9-fluorescent crRNA system for monitoring real-time simultaneous visualization of transcript RNA and genomic DNA in the live cell , Prevotella sp. (PspCas13b), and Ruminococcus flavefaciens (RfxCas13d) in Nicotiana benthamiana and target both the plant RNA viruses [tobacco rattle virus (TRV) and tobacco mosaic virus (TMV)] with appropriate crRNAs respectively. They found in transient assays LwaCas13a, PspCas13b, and CasRx variants arbitrate high interference activities against both the RNA viruses but CasRx founds to mediate more robust RNA interference activities against RNA viruses in both transient and stable overexpression assays as compared to other cas13 variants. Further, CasRx does not found to produce any collateral cleavage activity in planta and thus demonstrates strong specificity against the targeted virus and thus they conclude that CasRx is the most prominent endoRNase as compared to other Cas13 variants for RNA virus interference applications in planta  significantly and efficiently inhibited the HIV-1 replication when targeting four separate, non-overlapping sites in the HIV-1 transcript in all the cell line models as well in primary CD4+ T-cells. Therefore their study concluded that the use of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection will endow with an unconventional treatment modality against HIV  and Ruminococcus sp. (Rsp) and applied them to detect nucleic acid and they found that both the Cas13d exhibit efficient and rapid target RNA detection in picomolar range and even in the lower viral titer. On the whole, their study revealed that both EsCas13d and RspCas13d were able to detect the targeted RNA and even it can detect single-nucleotide variation with high sensitivity and specificity  in 30–45 min with high specificity and accuracy. Recently, Qiao et al., used two Cas13d effectors from cificity . TherefoThese experimental results represented that Cas13d (CasRx) can be used as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future diagnostic and therapeutic development.DD—has been fused to a variety of RNA-targeting modules, such as AD-gRNA, λN-peptide, SNAP-tag, cas13, etc.RNA editing is a post-transcriptional process that is broadly defined as any site-targeted alteration due to processes such as insertion and deletion of nucleotides, conversion of one base to another, splicing, etc. RNA-targeted genetic manipulation is capable of controlling target-protein functions, similar to genome editing, without the risk of damaging the original genomic information. Molecular tools to manipulate and measure RNA are limited. Though Small-interfering RNAs (siRNAs) and microRNAs (miRNA) can knock-down the RNA expression but its off-target effect is very much a matter of concern. In contrast to RNA-interference technology, a general RNA-mutagenesis technology enabling RNA modification is in its developing stage. In cellular machinery there are some enzymes, like two Adenosine Deaminases Acting on RNA (ADAR): ADAR1 and ADAR2, which convert adenosine to inosine (A → I) . InosineLeptotrichia wadei (LwaCas13a) is capable of providing comparable levels of knockdown as RNAi, but with dramatically improved specificity , comprising a 19 nucleotides-long anti-sense recruiting region (ARR) and a 49 nucleotides-long ADAR-recruiting region (APR), which directly induces A-to-I mutations by docking hADAR2 to the target-site . This wacificity .The above-mentioned targeted RNA editing tools, which rely on ectopic expression of exogenous editing enzymes, the delivery of exogenous proteins, or chemically modified guide RNAs, have many limitations including substantial global off-target editing of the genome and/or RNA transcripts, immunogenicity, oncogenicity, and delivery hurdles. To mitigate the problem Mali group has engineered ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing which recruited endogenous ADARs, and thus, vastly improved the durability of RNA editing. Two more RNA editing technologies, RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing)  and LEAPin vivo and human applications but recently some efforts have been made to increase the efficiency of the delivery which include methods such as Cas-inspired artificial CIRTS and nebulization; Moreover, immunogenicity, cytotoxicity, and stability of Cas13d components in the body fluids are the areas that still need to be addressed. Therefore, a better understanding of these processes can help scientists to develop engineered Cas13d nucleases with better efficacy and specificity. It is anticipated that Cas13d will be employed in various avenues but not limited to transcriptome editing. It will be more advanced than traditional CRISPRi approaches in terms of gene regulation. Finally, dCas13d tagged with fluorescence tags can help us visualize allele-specific RNA modifications like splicing, translation, polyadenylation, and degradation. To summarize, Cas13d is a “new molecular scissor” that demands more attention to fully unfold its potential and needs an ingenious solution to overcome the associated challenges to become a gold standard in transcriptome editing and application beyond RNA editing with salutary results in the coming future.It is undeniable that CRISPR will be an essential tool for genome and epigenome editing in the near future. Even though Cas9 stole all the limelight initially, it has limited therapeutic applications owing to its off-target effects. Further, the emergence of cas12 and Cas13 family nucleases, allowed the scientists to utilize CRISPR in several applications such as epigenetic modification, base editor, chromatin imaging, optogenetics, vesicle tracking, gene regulation, and the list is growing . Not onl


RNA stability plays an important role in gene expression. Here, using 3′ end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3′UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3′-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5′ splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity. APA is APA sites in the 3′-most exon lead mostly to isoforms with different 3′UTR sizes, whereas APA sites in introns change both the coding region and 3′UTR. Because of the central role of 3′UTR in mRNA metabolism , APA isoIn this study, by comparing poly(A)+ transcript abundance in newly made and pre-existing pools, we examine how APA isoforms differ in stability in multiple human cell lines. We examine how stability difference between isoforms varies across cell types and analyze the interplay between stability control and APA site choice. We further examine stability controls of lncRNAs and poly(A)+ RNAs encoded by the mitochondrial genome. Our results reveal 3′UTR features correlated with transcript stability differences between cell types and indicate potential contributions of RNA stability to neuronal cell identity.We hypothesized that transcript stability might be distinct in different cell types. To test this, we carried out metabolic labeling of cellular RNA with 4-thiouridine (4sU) in human HEK293T, HepG2, and SH-SY5Y cells . These c2(FT/4sU), named Stability Score (SS) for simplicity, to represent RNA stability , followed by separation of 4sU-labeled and non-labeled RNAs (see Methods for details). These two RNA pools, named 4sU labeled (4sU) and flow-through (FT), respectively, represent newly made and pre-existing RNAs, respectively. We subjected 4sU and FT RNA samples, as well as the total RNA, to the 3′READS+ method for 3′ end sequencing . The abutability . We founRequiring at least five reads in at least one sample as evidence of expression , we idenr = 0.88–0.91, Pearson Correlation, We first focused on mRNA genes, which accounted for the majority of poly(A)+ RNAs . Overallp < 0.05  line  and its Based on total RNA samples, we found that the transcripts with a higher abundance in SH-SY5Y cells compared to HEK293T cells tended to be upregulated in neurogenesis, whereas those with a lower abundance in SH-SY5Y cells compared to HEK293T cells tended to be downregulated in neurogenesis . This rep < 0.001, Wilcoxon test, p < 2.2 × 10−16, K-S test, Interestingly, we found that genes upregulated in neurogenesis had higher SS than those downregulated in neurogenesis in SH-SY5Y cells  between SH-SY5Y cells and HEK293T cells. The features we used included exonic and intronic sizes, splicing parameters, nucleotide contents of different regions, etc. see . Interesp < 2.2 × 10−16, K-S test, Because RNAs with higher GC contents could adopt more stable secondary structures than those with lower GC contents , we nextin vivo using dimethyl sulfate treatment followed by sequencing (DMS-seq)  . With DMr = 0.45, Pearson Correlation, MPPL2 and NUP98 are shown in MPPL2 had a larger ΔSS value in SH-SY5Y cells than HEK293T cells (2.5 vs. −1.6), NUP98 showed the opposite trend (−0.6 vs. −4.2). Interestingly, we found that GC-rich motifs were highly enriched in aUTRs of red genes  in all 3 cell lines were negative , indicatp < 0.05, Wilcoxon test, We next wanted to examine gene features related to IPA isoform stability as we did with 3′UTR isoforms. However, because our 3′READS + reads for IPA transcripts were largely located in the middle of an intron, we could not accurately derive sequence features, such as CDS size, 3′UTR size, etc., for most IPA isoforms. We therefore focused on the introns in which IPA sites were identified, including intron size, 5′ splice site (5′SS) strength, and 3′ splice site (3′SS) strength. Splice site strengths were based on the maximum entropy (MaxEnt) method . We divip < 0.05, Wilcoxon test, p > 0.05, Wilcoxon test, In addition to protein-coding transcripts, our 3′READS + data identified many lncRNAs, including canonical lncRNAs, p-lncRNAs and eRNAs, RNAs encoded by the mitochondrial genome (named mtRNAs), pseudogene transcripts, and unannotated intergenic RNAs . We calcWe next further examined mtRNA genes, which included 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes . We founIn this study, we systematically studied poly(A)+ RNA stability in three human cell lines, namely, HEK293T, HepG2, and SH-SY5Y. We found that while mRNA stability controls are generally similar in these cells, certain transcripts display differential stability levels across the cell lines, especially in SH-SY5Y cells. We show that GC content and RNA secondary structures of transcripts correlate with distinct mRNA stability values in SH-SY5Y cells, which also impact 3′UTR isoform stability differences in these cells. Given the relevance of our SH-SY5Y versus HEK293T comparison to neurogenesis in gene expression and APA regulation, we conclude that modulation of mRNA stability could contribute to establishment of neuronal cell identity.Our RNA stability analysis is based on comparison of transcript abundances in newly made versus pre-existing RNA pools. We show that the Stability Score (SS) derived from this comparison are well correlated with RNA decay rates that were measured by using transcriptional shutdown. As such, our method offers a simple and meaningful approach to examine RNA stability. On the other hand, it is worth noting that the accuracy of SS could be compromised if there are alternations of transcription during the course of metabolic labeling. In other words, when newly made and pre-existing RNAs are produced in distinct transcriptional contexts, our method would be affected by large noises. However, we do not expect this to be an issue when cells are under normal growth conditions, as in this study.We show that GC content and RNA structures could impact stability differences between 3′UTR isoforms, corroborating the gene-to-gene analysis result. It is worth noting that APA isoforms that differ in 3′UTRs offer an accurate and efficient approach to examine the impact of 3′UTR on mRNA metabolism. This is because the 3′UTR isoforms presumably have identical sequences except for their aUTRs; their comparison, therefore, addresses potential, confounding features in CDS and 5′UTR. The underlying mechanism(s) for RNA stability control via GC content and RNA secondary structures, however, is unclear. There are a group of RNA-binding proteins that specifically interact with sequence motifs with high GC contents  and/or Rper se. Therefore, subtle differences in half-life values could be significant in this work but not in the Spies et al. study. Nevertheless, our comparative approach using multiple cell lines enabled us to identify cell type-specific differences in RNA stability, mitigating any intrinsic biases in using SS to measure stability.Our finding that long 3′UTR isoforms are generally less stable than short 3′UTR isoforms is largely in line with our previous studies . NotablyWe found that IPA isoforms are generally less stable than isoforms using 3′-most exon PASs. This result suggests that while some IPA isoforms could diversify the protein-coding potential of genes, most IPA events may lead to unstable RNAs that do not have substantial impacts on cell functions. In other words, IPA may be employed mainly to downregulate gene expression. Interestingly, we found that both intron size and 5′SS strength play negative roles in IPA isoform stability. 5′SS strength may be related to nuclear retention of IPA transcripts through U1 snRNP, as shown recently for lncRNAs . The negOur data indicate that isoform stability variation counters APA site choice, i.e., the isoforms preferentially expressed in 1 cell type are also less stable in this cell type, as compared to other cell types. Interestingly, this trend applies to both 3′UTR APA isoforms and IPA isoforms, indicating that stability regulation of a specific transcript is connected to its 3′ end processing activity. Notably, a recent study by the Struhl lab reported a similar compensatory link between transcript decay and its 3′ end processing in yeast . This phSeveral types of lncRNAs, including regular lncRNAs and p-lncRNAs, were found to be generally more stable in SH-SY5Y cells compared to HEK293T or HepG2 cells. Regular lncRNAs are stand-alone genes, similar to mRNAs. p-lncRNAs are associated with gene promoters, also known as upstream antisense RNAs or PROMPTs . Both RNOur finding that mitochondrial RNAs are more stable in SH-SY5Y cells than other 2 cell lines may have important implications for neurobiology. Mitochondrial dysfunctions are known to be involved in the pathogenesis of several neurodegenerative disorders , highlig4 cells/cm2 onto matrigel-coated chamber-slides in the presence of 20 ng/ml FGF2. After 72 h, cultures were shifted to NS-A Basal medium containing 2% FBS and were grown for another 2 weeks. Neurons were harvested by using accutane to dissociate cells, and RNA was extracted by using Trizol.Human HEK293T and HepG2 cells were cultured in high glucose DMEM with 10% fetal bovine serum (FBS). SH-SY5Y cells were cultured in DMEM/F12 with 10% FBS, 1% sodium pyruvate, and 1% glutamate. All media contained 1% Penicillin/Streptomycin solution (Sigma). Human neural stem cells (NSCs) were obtained from Vescovi et al. . NSCs weCells at ∼70% confluency were used for metabolic labeling of RNA as previously described . Briefly25 magnetic beads (NEB) and was fragmented on-bead by RNase III (NEB). After washing away free RNA fragments, poly(A)+ RNA fragments were eluted from the beads and precipitated with ethanol, followed by ligation to heat-denatured 5′ adapter (5′-CCUUGGCACCCGAGAAUUCCANNNN) with T4 RNA ligase 1 (NEB). The ligated products were captured by biotin-T15-(+TT)5  bound to Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). After washing, RNA fragments on the beads were digested with RNase H and then eluted from the beads. After precipitation with ethanol, RNA fragments were ligated to a 5′ adenylated 3′ adapter (5′-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC (Bioo Scientific) with T4 RNA ligase 2 . The ligation products were then reverse transcribed by using M-MLV reverse transcriptase (Promega), followed by PCR amplification with Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers for 12–18 cycles. PCR products were size selected twice with AMPure XP beads (Beckman Coulter). The size and quantity of the cDNA libraries were examined on an Agilent Bioanalyzer and sequenced on an Illumina HiSeq machine (1 × 150 bases).The 3′READS+ procedure was carried out as previously described . Briefly3′READS+ data were analyzed as previously described . The 5′ 2 (RPM of FT sample/RPM of 4sU sample) value is called Stability Score (SS). Two biological replicates were averaged. Scaled mRNA decay rate data was obtained from a previous study  in flow-through (FT) sample and 4sU sample were calculated and normalized by using the DESeq method. The logus study , which i. Differential expression of proximal PAS and distal PAS isoforms was carried out by using DEXSeq  and relative abundance difference >5%. Relative expression (RE) of the two isoforms was calculated by log2 . aUTR size was the distance between the proximal and distal PASs in the 3′UTR.For 3′UTR isoform analysis, the two APA isoforms containing 3′-most exon PASs with the highest expression levels were selectedg DEXSeq . Signifip-values to indicate significance of association between a gene set and a GO term. GO terms associated with more than 1,000 genes were considered too generic and were discarded. To reduce redundancy, any GO term that overlapped with a more significant term by >90% was removed.Gene ontology analysis was carried out by using the GOstats package in R . The Fisr value and individual and cumulative R2-value were calculated using the “cor” and “lm” functions in R. The PAS count of a gene and conservation of PAS were obtained from PolyA_DB v3 .Nucleotide content in a specific region was calculated by using the BSGenome package in R. Hexamer frequencies in 3′UTRs were calculated by using the Biostrings package in R and were compared between gene sets. The RNAfold function of the ViennaRNA package  was used


Extant biology uses RNA to record genetic information and proteins to execute biochemical functions. Nucleotides are translated into amino acids via transfer RNA in the central dogma. tRNA is essential in translation as it connects the codon and the cognate amino acid. To reveal how the translation emerged in the prebiotic context, we start with the structure and dissection of tRNA, followed by the theory and hypothesis of tRNA and amino acid recognition. Last, we review how amino acids assemble on the tRNA and further form peptides. Understanding the origin of life will also promote our knowledge of artificial living systems. Among the three steps in the central dogma, translation is the most important as it bridges the world of nucleic acids and the world of amino acids. In the “RNA world” hypothesis , RNA camDNA self-copy, i.e., replication, and DNA-templated RNA polymerization, i.e., transcription, are more intuitive and practicable in prebiotic settings compared to RNA-coded peptide formation. Nowadays, translation takes place in the endoplasmic reticulum membrane in the cytoplasm with messenger RNA as the template, transfer RNA as the adaptor, and ribosome RNA as the catalytic core. How tRNA and mRNA formed and how primal peptides were coded in the prebiotic era without auxiliary proteins are interesting questions. Although these questions were posted decades ago and various theories have been put forward, great attention and practical effort have been given to these issues in recent years. Here, we summarize the hypotheses and experimental milestones regarding this question. We focus on tRNA evolution, codon assignment, and coded peptide formation, and provide our ideas on the origin of translation.Transfer RNA, the core of genetic coding, is an invaluable molecular fossil that can help in discovering the origin of translation. After revealing the double helix structure of DNA in 1953, Crick  proposedIn extant life, the DNA sequence that codes the tRNA is either separated with introns, fragmented, or rearranged . This isPast decades have witnessed significant progress focusing on the stepwise assembly of tRNA. In the beginning, Woese  posited At the same time, Di Giulio ,27 modifContrary to the above models, Maizels and Weiner  disassemSeveral further ideas have been put forward along with the models above, including the tri-minihelices model ,36, the Before discussing the onset of RNA-coded peptides, the emergence of RNA and amino acid precursors in the prebiotic settings needs to be clarified. In the Miller experiment, various products were synthesized and identified under a possible primitive condition. Gly, Ala, and β-Ala were identified at the 100 µM scale. Asp, Glu, Ser, and Val were observed as well, albeit in a lower concentration ,42,43. LDue to the sequential emergence of amino acids, the assignment of genetic codes must be stepwise. The genetic codes are universal in the three kingdoms; therefore, the assignment of amino acids to the 64 triplets must be established before the division of the kingdoms. In parallel with the evolution of tRNA, several hypotheses for codon assignment have been proposed in the past decades, among which the frozen accident theory and stereochemical theory were first. Frozen accident theory claimed that codons were randomly assigned and are impossible to change significantly afterwards ,51. The Stereochemical theory proposed a different viewpoint: that the assignment was rational because of the stereochemical interactions, or the physical affinity, between the amino acids and nucleotides. The initial attempt to interpret the interaction, which dated back to 1954, was the diamond code theory, which proposed that a ‘key-and-lock’ relationship existed between a specific amino acid and the rhomb-shaped ‘holes’ in the DNA strand ,57,58,59E. coli tRNAAla eliminated aminoacylation both in vivo and in vitro  an an96] anThe second step of translation is peptidyl transfer, which is assisted by the ribosome; this is one of the core processes in extant biology. In all three kingdoms of life, the ribosome catalyzes the peptidyl transfer and monitors the accuracy of the mRNA-templated translation. Dissection of ribosomes enlightens our understanding of translation in prebiotic times. The prokaryotic ribosome consists of two separate components, the SSU and the LSU a. The SSLSU is divided into six domains based on structure and function, among which domain V accounts for peptidyl transferase activity . InteracSeveral mimicked or minimized ribosome PTCs have been designed and tested. A selected PTC-like ribozyme suggested that short, folded oligoribonucleotides could aid RNA–RNA interaction and peptidyl transfer, further proving the idea that the LSU originated from a short RNA . Based oWe have reviewed here three major questions about the origin of translation: tRNA evolution, codon assignment, and coded peptide formation. The two halves model from Maizels and Weiner indicates that the acceptor stem domain is ancient and ligates with the anticodon loop domain. The frozen accident theory would be correct if the fusion of the two domains were random. The stereochemical theory would also be correct if sequence-dependent aminoacyl transfer was discovered. Coded peptide formation without previously synthesized peptides should have been possible when tRNAs were juxtaposed with an ssRNA with the help of ribozymes. Other topics of concern, e.g., amino acids activation, random peptide synthesis, etc., may also be interesting but are not covered here.Numerous issues remain unsolved or have not even been investigated. To the best of our knowledge, no research has shown whether mRNA or PTC came first. If peptides can arise from anticodon loops and ssRNA complexes without PTC, then mRNA must have come earlier than the ribosome. The ssRNA, which acted as the extant mRNA, recorded the peptide sequence. To create positive feedback, the initial peptides must take on the function to promote the RNA loop and ssRNA interaction and accelerate peptidyl transfer. However, if a peptide could be formed without ssRNA but needs a ribozyme, then mRNA must come later than PTC or the proto-ribosome. Although the peptides could assist aminoacylation and peptidyl transfer, how could the sequence information in the peptide be recorded ? This puExtant prokaryotic translation starts with N-formylmethionine (fMet). The formyl group at the amino end of a dipeptide prevents it from cyclizing to 2,5-diketopiperazine (DKP), which is highly stable and cannot be linearized. Therefore, the formyl protecting group is a good strategy to preserve peptidyl transfer products; however, did the N-formyl amino acid present prior to acylation, or did free aminoacylated tRNA arise first then the formyl group came to cap the N-terminus? Did the chemistry for recycling DKP already exist in the Darwin Pond, or did the DKP never bother the peptide formation? This is currently unknown.As is known, peptides can form spontaneously in the prebiotic context ,121. TheCompartmentalization is requisite for a living system. Did the compartment arise after the translation was established, or did they emerge at the same time? Would the compartment benefit the translation efficiency and fidelity? How did the translation products trigger the Darwinian evolution? These intriguing and challenging questions are left open for further study.Investigating the origin of translation not only provides us with the knowledge of the origin of life and satisfies our curiosity about abiogenesis, but also enables us to build non-canonical and functional biopolymers in a cell-free medium or protocells. Knowledge of the translation machine, including the tRNA and rRNA, helps us to develop new therapies on the protein expression level. Recent advances in suppressing premature termination codons with anticodon-engineered tRNA are encouraging steps to conquer single-gene disorders using non-canonical tRNA ,128,129.


High resolution manometry (HRM) is the gold standard for diagnosis of esophageal motility disorders. This is traditionally conducted without sedation, using intranasal intubation with a manometry probe. Inserting the HRM probe immediately after endoscopic examination allows for endoscopic visualization and sedation for probe placement. This method is used commonly used for our patients who are post-peroral endoscopic myotomy (POEM). However, it is unclear whether short acting sedative drugs administered during endoscopy can affect contractility measurements during HRM.The purpose of this study was to determine if conscious sedation with short-acting anaesthetic agents (Midazolam and Fentanyl) influenced HRM readings in patients undergoing evaluation post-perioral endoscopic myotomy (POEM).We conducted a retrospective cohort study, comparing patients post-POEM who had endoscopy under conscious sedation with placement of the manometry probe versus those without preceeding endoscopy/sedation. Post-POEM patients who had undergone HRM over a 3-year period (2019-2022) were identified from motility lab records. Electronic charts were reviewed for data abstraction, and HRM parameters  were determined for both groups and compared using t-tests, and error listed as standard error of the mean.From 2019-2022, 15 patients underwent manometry using a non-endoscopic approach with no sedation and 25 underwent manometry following endoscopy with conscious sedation and placement of the probe during the procedure. In the latter group, HRM occurred within 2 hours of probe placement. The mean doses of fentanyl and midazolam were 115mcg (+/-29.7) and 3.3mg (+/-1.3) respectively. The mean end-expiratory lower esophageal sphincter pressure was 9.8 mmHg (+/- 2.9) in the non-endoscopic group and 26.24mmHg (+/- 5) in the endoscopy group (p <0.02). The median integrated relaxation pressure was 11.2 mmHg (+/-1.2) in the non-endoscopic group and 14.52mmHg (+/- 0.9) in those in the endoscopy group (p <0.04).In conclusion, this small, retrospective cohort study shows that manometry probe placement after endoscopy with conscious sedation may affect esophgeal HRM measurement parameters in the post-POEM patient population. Physicians should consider delaying HRM measurements for >4 hours to maximize clearance of the sedative medications.NoneNone Declared


Proximity labeling employs modified biotin ligases or peroxidases that produce reactive radicals to covalently label proximate proteins with biotin in living cells. The resulting biotinylated proteins can then be isolated and identified. A combination of programmable DNA targeting and proximity labeling that maps proteomic landscape at DNA elements with dCas9-APEX2 has been established in living cells. However, defining interactome at RNA elements has lagged behind. In combination with RNA-targeting CRISPR-Cas13, proximity labeling can also be used to identify proteins that interact with specific RNA elements in living cells. From this viewpoint, we briefly summarize the latest advances in CRISPR-guided proximity labeling in studying RNA–protein interactions, and we propose applying the most recent engineered proximity-labeling enzymes to study RNA-centric interactions in the future. RNA–protein interactions are pervasive in regulatory RNAs and RNA-mediated processes in cells ,2,3. A gRecently, researchers have applied the RNA-targeting CRISPR-Cas13 system to proximity labeling to achieve programmable RNA-interacting proteins biotinylation ,12,13,14Taken together, these new methods expedite the analysis of RNA interactome in living cells. Compared with previous RBPL or RaPID labeling, CRISPR-guided proximity labeling method is programmable and easy-to-use, which enables arbitrary RNA targeting and the biotinylation of RNA-associated proteins. Moreover, researchers can build inducible CRISPR-guided proximity labeling enzyme fusion cell lines, allowing the spatiotemporal control of proximity labeling activity and the mitigation of high background of biotinylation. CRISPR-guided proximity labeling methods are usually validated with well-known RNA–protein interactions first and then the unknown RNA interactome was explored with mass spectrometry to expedite the analysis of endogenous RNA–protein interactions. Similarly, Zhang et al. reported a new proximity labeling system by using PUT-IT to label proximate proteins of specific RNAs . HoweverAlthough bioID and APEX2 are useful tools for proximity labeling, they have some common drawbacks. For example, bioID takes 18~24 h of reaction time to obtain enough labeled materials for quantitative proteomics ,20. SuchE. coli birA-derived enzyme for protein–protein interaction detection [Recently, Kohki et al. reported AirID, an etection . In thisetection . UltraIDetection , was defAlthough numbers of methods have been developed to probe RNA activity, improving RNA-centric targeting affinity and sensitivity is still in early development. It is also expected that a programmable RNA-targeting system could broadly target cellular low-abundance transcripts. The combination of evolved proximity-labeling enzymes and RNA-targeting technology would further expedite the functional analysis of endogenously expressed RNAs in the future.The proximity labeling method employs promiscuous biotin ligases or peroxidases to label proximate proteins. This method is most commonly used to probe protein–protein interactions and the constituents of subcellular compartments refractory to biochemical isolation ,25. HoweProximity labeling is particularly suitable for probing insoluble or inaccessible subcellular molecules and for detecting weak or transient intermolecular interactions ,22,23,25


As a reaction to environmental changes, m6A modification showed a high degree of sensitivity and complexity. We investigated differences in gene medleys between dark-induced leaf senescence (DILS) and developmental leaf senescence in barley, including inter alia RNA modifications active in DILS. The identified upregulated genes in DILS include RNA methyltransferases of different RNA types, embracing enzymes modifying mRNA, tRNA, and rRNA. We have defined a decisive moment in the DILS model which determines the point of no return, but the mechanism of its control is yet to be uncovered. This indicates the possibility of an unknown additional switch between cell survival and cell death. Discoveries of m6A RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in metabolic reprogramming. Nonetheless, there is no such data about the process of leaf senescence in plants. In this scope, the prospect of finding connections between the process of senescence and m6A modification of RNA in plants seems to be compelling.Crop losses caused by climate change and various (a)biotic stressors negatively affect agriculture and crop production. Therefore, it is vital to develop a proper understanding of the complex response(s) to (a)biotic stresses and delineate them for each crop plant as a means to enable translational research. In plants, the improvement of crop quality by m One example is epitranscriptomics, defined as posttranscriptional modifications of RNA bases that alter gene expression without modifying the RNA sequence and regulate the stability and transport of the RNA.6-methyladenosine (m6A) is the most frequently occurring and conserved internal modification of eukaryotic RNA  (This perspective article synthesizes the knowledge in the scope of epigenetic regulation of induced leaf senescence based on the modification of ml (DILS)  which de2As per the current state of knowledge, senescence is a developmental stage that: (i) is an event of transient differentiation after growth; (ii) might or might not be followed by death; and (iii) is wholly dependent on the viability of cells and a particular gene expression . SenescePlants’ growth is greatly supported by leaves, which are the best organs for using light energy and producing additional photosynthates with simultaneous minimization of total anabolic cost. When under stress, it may be advantageous for the plant if a leaf that is not photosynthetically active passes through senescence and makes its resources available to other organs. Senescence induction needs to be tightly managed in order to prevent activation that only occurs under short-term adverse situations. Light is essential for controlling the senescence of leaves, which is consistent with the significance of leaves in photosynthesis. Many plant species experience fast senescence as a result of light deprivation, especially when the plant is only partially impacted  events . It is nIn the field of gene expression regulators, specifically RNA chemical changes, new players have recently surfaced. RNA modifications were first discussed in the 1950s , but rec6-methyladenosine, is thought to be the most prevalent, reversible epitranscriptomic mark, functionally active in both animal and plant RNAs. It occurs when a methyl group is attached to the nitrogen-containing base at the sixth position of the adenine residue of RNA , maintenance of circadian and seasonal plant rhythms, and late flowering  is a model organism both genetically and genomically: it is characterized by a high degree of natural variation and by the adaptability to numerous different cultivation environments. Its simple diploid genome has already been sequenced (Gramineae species) following maize, wheat, and rice when it comes to global production (https://www.statista.com). As a result, barley stands out from other crop plants because of its crucial role in both agriculture and science. Advances in both aspects create a positive feedback loop, which allows the barley to take a leading role in meeting the great challenges of climate change, as well as the growth of the human population.We must further our understanding of the epigenetic regulators in crops during senescence brought on by stress and their basic molecular mechanism. Investigating nescence . Nonetheequenced  and the oduction . The vol6A modifications in the aging of animal models as well as cell senescence has been previously reported  have shown that genes connected to plant defense are characterized by significant changes in their m6A transcript level during the response to the wheat yellow mosaic virus (WYMV)  . In conts (WYMV)  the noveo fruits . Moreoveadaption .6A 2Target, MeTDiff), in contrast with plant RNA modifications, which can be found only in one database: PRMdb , including RNA transcribed from transposable elements (repeat RNA) (6A was shown to stimulate the degradation of a subset of repeat RNA species through nuclear exosome-targeting-mediated nuclear degradation (In consideration of the fact that RNA modification meat RNA) , the bareat RNA) . Also, mradation .6A demethylation and originally identified as a fat mass- and obesity-associated protein , involving human FTO – an enzyme mediating RNA m protein  – which  protein . Accordi6A RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in the metabolic reprogramming of leaf cells between strategies of cell survival and cell death.Senescing cells must retain their ability to maintain homeostasis at each stage, which is why the effectiveness of the senescence process’ regulation is a sign of the vitality of the senescing cells. We have defined a decisive moment in the DILS model  which de6A modification are thought to be caused by a dynamic interplay between RNA methyltransferases, RNA demethylases, and proteins interacting with modified RNA . All of these stressors individually impose unique complexity on a plant’s biological and genetic response, as well as on solutions to reduce them. In order to facilitate translational research, it is crucial to have a thorough understanding of the diverse response(s) to abiotic stresses and define them for each agricultural plant. Numerous reviews have concentrated on several aspects of plant abiotic stresses and outlined the need to define the processes that occur as well as possible mitigation strategies, such as the use of “antistress effectors,” the development of novel genetic approaches, and the development of new resilient crops. Decoding the molecular mechanics of development and stress resistance is crucial for better crop management, increasing barley production and quality. In plants, the improvement of crop quality by m6A editing is believed to be a promising strategy. The m6A modification demonstrated a high level of sensitivity and complexity in response to environmental changes. Importantly, crop resistance may be improved by editing m6A modifications on transcripts relevant to plant resistance . There aPublicly available datasets were analyzed in this study. This data can be found here: GenBank (RRID: SCR_002760), Ensembl (RRID: SCR_002344).ES-N conceived the topic of the manuscript. ER, MG, MA-J, JM, UT and ES-N wrote the manuscript. MG prepared the figure. ER was responsible for the layout of the manuscript and prepared the table and the data contained therein. ES-N coordinated the writing of the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


RNA interference (RNAi) is a powerful tool to control the expression of a target gene, yet RNAi triggers, typically small interfering (si) or short hairpin (sh) RNAs, are often prone to inducing adverse side effects that can limit their therapeutic index. By embedding Argonaute 2 (Ago2)-dependent shRNAs (agshRNAs) within a micro (mi)RNA backbone, and by thus enabling tissue-specific expression from an RNA polymerase (pol) II promoter, Alsing and colleagues have now taken a pivotal step at increasing the specificity and safety of therapeutic RNAi. Additionally, an RNA-seq-based comparison permitted the authors to identify side effects of agshRNAs or their conventional shRNA counterparts. Thereby, this study concurrently and compellingly demonstrates the potential to improve RNAi safety and efficiency, as well as the power of assays to monitor sequence-specific or -unspecific side effects. At the same time, this work highlights that the therapeutic window ensuring sufficient and concomitantly safe target gene knockdown may be narrow.in vivo toxicity.The concept to harness the endogenous RNAi pathway for targeted mRNA silencing by exogenous introduction or expression of artificial RNAi triggers had been established over 2 decades ago. Among the most frequently employed RNAi triggers are shRNAs, which, especially when delivered by lentiviral (LV) or adeno-associated viral vectors, can achieve sustained target knockdowns. Their expression is usually driven by RNA pol III promoters such as U6, which can produce large quantities of RNAi effectors in transduced cells and mediate robust knockdowns. However, in a manner depending on shRNA sequence, abundancy, and other parameters, shRNAs can also induce adverse side effects comprising (1) off-targeting, (2) saturation of the cellular RNAi machinery and competition with endogenous miRNAs, and (3) induction of immune responses, in the worst case culminating in Vegfa-targeting shRNAs as well as corresponding agshRNAs based on miR451 or miR324. As hoped for, in cultured cells, the agshRNAs showed (1) no detectable passenger-strand activity against an antisense target, (2) decreased competition with endogenous miRNAs, and (3) partially reduced toxicity after LV transduction compared to their shRNA counterparts. Both, competition and toxicity, were more strongly influenced by the specific target sequence than by the structure of the RNAi effector. Interestingly, both side effects were abolished when the expression of agshRNAs was switched from the U6 to an RNA pol II promoter, by embedding agshRNAs in a miRNA backbone (miR-agshRNAs). Alas, this came at the cost of a decrease in knockdown efficiency. To demonstrate the in vivo applicability of miR-agshRNAs, Alsing et al. chose the RPE-specific VMD2 promoter they had used for miRNA-based knockdown of Vegfa before.,Vegfa in transduced compared to non-transduced cells. However, the overall transduction efficiency was too low to allow detection of an effector-induced knockdown without a prior fluorescence-activated cell sorting-based isolation of transduced cells.In their current study, Alsing and colleagues generated and directly compared To moreover study the impact on the cellular transcriptome, Alsing et al. transduced ARPE19 cells with LV vectors containing U6 promoter-driven expression cassettes for three corresponding shRNA/agshRNA pairs. Using RNAseq combined with clustering of differentially regulated genes, this enabled the identification of several side effects elicited by the different effectors through gene ontology enrichment and heptamer seed match analyses. While these results further exemplify the benefits of agshRNAs, such as reduced off-target knockdowns mediated by the passenger-strand seed, they also revealed sequence-specific off-target effects that are shared between sh- and agshRNAs with a common guide strand.in vivo toxicity, followed by (2) agshRNAs with reduced passenger-strand activity and endogenous miRNA competition, the new class of (3) miRNA-embedded agshRNAs driven from tissue-specific RNA pol II promoters reported here promises yet another leap in RNAi specificity and safety  classical U6 promoter-driven shRNAs and their propensity for d safety . Still,


Caenorhabditis elegans, in which their axons and synapses overlap minimally with their neighboring neurons within the same class. We found that while EGL-20/Wnt controls axonal tiling, their presynaptic tiling is mediated by a gap junction protein UNC-9/Innexin, that is localized at the presynaptic tiling border between neighboring dorsal D-type GABAergic motor neurons. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in controlling tiled presynaptic patterning. While gap junctions are crucial for the proper functioning of the nervous system as channels, our finding uncovered the novel channel-independent role of UNC-9 in synapse patterning.Precise synaptic connection of neurons with their targets is essential for the proper functioning of the nervous system. A plethora of signaling pathways act in concert to mediate the precise spatial arrangement of synaptic connections. Here we show a novel role for a gap junction protein in controlling tiled synaptic arrangement in the GABAergic motor neurons in  Drosophila, L1 lamina neuron axons are arranged in columns in the medulla such that they only form synaptic connections within a single column in a non-redundant manner  motor neurons form en passant cholinergic chemical synapses onto the dorsal body wall muscles in a way that each presynaptic domain from a single DA neuron does not overlap with those from the neighboring DA neurons  in mammals and innexins (INX) in invertebrates . While ttivities . Mammalitivities . Howevertivities . For exativities . A PANX1tivities . Upregultivities . Functiotivities .C. elegans nervous system revealed axonal and dendritic tiling of the dorsal and ventral D-type GABAergic motor neurons (DDs and VDs)  reconstruction of the and VDs) . Here ween passant synapses along their axons onto the dorsal body wall muscles and the VD motor neurons (Cell bodies of six DD class of GABAergic motor neurons (DD1–DD6) reside in the ventral nerve cord . Each DD neurons . Previou neurons . As a reflp-13), and the membrane-associated mCherry (mCherry::CAAX) under the DD6 neuron-specific promoter (Pplx-2)   under the DD neuron-specific promoter (Pmethods) . The flp neurons , which c neurons . To visuervation .Together, we showed for the first time, that DD5 and DD6 have tiled axons, dendrites, and presynaptic patterning in live animals, which are in agreement with the previous EM reconstruction data .C. elegans  in C. elegans, egl-20 is expressed in the cells around preanal ganglions (egl-20(n585), which carries a missense mutation in one of the conserved cysteine residues, we observed overextension of the DD5 posterior axon (egl-20(n585) mutant (egl-20(n585) mutant  mutant , suggest) mutant . We couldendrite . From thegl-20(n585) mutant, we asked whether their presynaptic patterns are also compromised. If the presynaptic tiling between DD5 and DD6 is simply a consequence of axonal tiling, synapses will form throughout the DD5 axon in the egl-20(n585) mutants, resulting in significant overlap between the synaptic domains of DD5 and DD6. Surprisingly, despite the significant axonal overlap between DD5 and DD6, we observed little defect in the presynaptic tiling pattern (egl-20(n585) mutant compared with wild type, the small degree of presynaptic tiling defect did not reflect the large axonal overlap between DD5 and DD6 (Since DD5 and DD6 axons overlap in the  pattern . While t and DD6 . This obegl-20(n585) mutant, we next examined the postsynaptic dendritic spine patterning of DD5 using a transgenic strain that expresses ACR-12::GFP under the flp-13 promoter  mutant, the postsynaptic domain within the DD5 posterior dendrite is significantly longer compared with wild type and is comparable to the length of the DD5 posterior dendrite  mutant, the length of the DD5 postsynaptic domain is determined by the length of DD5 posterior dendrite.As the DD5 posterior dendrite also overextends in the promoter  ; egl-20(n585), egl-20(n585); rap-2(gk11), and egl-20(n585); mig-15(rh148)  and unc-9(e101 or tm5479) null mutants exhibited a significant presynaptic tiling defect (egl-20(n585); unc-9(e101) mutants is fully rescued by the Pegl-20::egl-20 transgene, which rescues the axonal tiling defect (unc-9 in presynaptic patterning (unc-9(e101) single mutants did not exhibit presynaptic tiling defect due to the normal axonal tiling in the presence of egl-20  mutants (egl-20(n585); unc-9(e101) double mutants is not due to an increased axonal tiling defect.We next sought to identify genes that are responsible for presynaptic tiling between DD5 and DD6 neurons. Previously we showed that Semaphorin-Plexin signaling mediated by PLX-1/Plexin, RAP-2/Rap2A, and MIG-15/TNIK is crucial for the presynaptic tiling of two DA-class cholinergic motor neurons . However5(rh148) , suggest5(rh148) . These g5(rh148) . Given tnd unc-9 . Among tg defect . Ectopicg defect . The preg defect , suggesttterning . Consistf egl-20 . Importa mutants . Therefoegl-20(n585); unc-9(e101) mutants represent bona fide synapses, we examined co-localization between mCherry::RAB-3 puncta in the DD5 neuron and NLG-1/Neuroligin, which is localized at the postsynaptic site of the GABAergic neuromuscular junctions (egl-20(n585); unc-9(e101) mutants are apposed by NLG-1::AID::GFP, similar to those in wild type (egl-20(n585); unc-9(e101) double mutant (egl-20(n585); unc-9(e101) double mutant likely represent bona fide synapses.To determine if the ectopic RAB-3 puncta present in the DD5 posterior axon of unctions , using eunctions . We obseild type . The mosild type . This isild type  and 2E (e mutant . These oegl-20(n585) mutant is present at the second larval (L2) stage (egl-20(n585); unc-9(e101) mutant  stage, when the dorsal neurites switch their fate from dendrite to axon . We foun2) stage . We obse) mutant . As the unc-9 is also required for the maintenance of presynaptic tiling, we conducted rescue experiments using a heat shock promoter. We found that the heat-shock-induced expression of unc-9 at the L2 stage after presynaptic tiling is established did not rescue the presynaptic tiling defects in egl-20(n585); unc-9(e101). This result is consistent with the idea that unc-9 is required for the establishment of the presynaptic tiling of DD5 and DD6 (To determine if  and DD6 .7×gfp11) at the C-terminus of endogenous locus of unc-9 using CRISPR/Cas9 genome editing. The unc-9(miz81 [unc-9::7×gfp11]) animals exhibit uncoordinated locomotion pattern, probably because UNC-9::7×GFP11 is expected to form constitutively open gap junction channels (see below). We then expressed the remaining part of GFP (GFP1-10) specifically in DD neurons using the flp-13 promoter to reconstitute the fluorescent UNC-9::7×GFP exclusively in the DD neurons. In wild type animals, UNC-9::7×GFP is localized at the anterior tip of the DD6 axon and dendrite  mutant, in which DD5 axon overextends to the DD6 axonal region, UNC-9::7×GFP was localized at the anterior tip of DD6 axon and dendrite similar to wild type, even though the signal intensity of the UNC-9::7×GFP at the tip of DD6 axon was slightly reduced (egl-20(n585) single mutant  single mutant, the UNC-9 localization at the presynaptic tiling border in egl-20(n585) mutant suggests that UNC-9 functions as a positional cue to define the presynaptic tiling border between DD5 and DD6 in egl-20 mutant background. While the UNC-9::7×GFP localization appears to be unaffected in the dendrite, the DD5 posterior postsynaptic domain, judged by ACR-12::GFP, is significantly longer in the egl-20(n585) single mutant  mutant did not exhibit a presynaptic tiling defect between DA8 and DA9  and a tight junction associated protein ZO-1  and zoo-1(tm4133) null mutants (zoo-1(tm4133) mutant (egl-20(n585); nlr-1(gk366849) and egl-20(n585); zoo-1(tm4133) mutants (unc-104(e1265), in which axonal transport is severely compromised (unc-104(e1265) mutant, due to a severe defect in axonal transport of presynaptic materials.Recent work in ein ZO-1 . In the on NLR-1 . In zebron NLR-1 . While b neurons , the loc mutants , suggest) mutant . Consist mutants . We alsopromised . We founpromised . These runc-9 is expressed broadly in the nervous system and non-neuronal tissues, including the DD neurons and their postsynaptic body wall muscles (unc-9 under the rgef-1 promoter rescued the presynaptic tiling defect of the egl-20(n585); unc-9(e101) double mutants  phenotype similar to unc-9 mutants (egl-20(n585); unc-1(e719) double mutants. Interestingly, unlike egl-20(n585); unc-9(e101) double mutants, egl-20(n585); unc-1(e719) double mutants did not exhibit a presynaptic tiling defect ; unc-1(e719) mutants, egl-20(n585) unc-24(miz225) double mutants and egl-20(n585) unc-24(miz226); unc-1(e719) triple mutants did not exhibit a presynaptic tiling defect  control border cell migration independent of their gap junction channel activity  can rescue the presynaptic tiling defect by expressing it under the rgef-1 pan-neuronal promoter or under the flp-13 DD-neuron-specific promoter. Surprisingly, both pan-neuronal and DD neuron-specific expression of unc-9(Δ18N) rescued the presynaptic tiling defect of egl-20(n585); unc-9(e101) mutants (unc-9 but not unc-9(ΔN18) rescued the kinker phenotype of the unc-9(e101) mutant (unc-9 mutants are largely due to the defective gap junction channel activity (unc-9 mutants by unc-9(Δ18N) strongly suggests that UNC-9(ΔN18) forms a defective, likely a constitutively closed, gap junction channel.While Cxs and INXs function primarily as gap junction channels, recent studies have revealed channel-independent roles for gap junction proteins . For exaactivity . Cx43 anactivity . Given tchannels . In an achannels . This re mutants . Importa) mutant . As the activity , the faiunc-9(e101) mutant. In these experiments, we expressed either wild type unc-9 or unc-9(ΔN18) in the muscle cells using the muscle-specific myo-3 promoter (Pmyo-3) in the unc-9(e101) mutant background. Cytoplasmic GFP was co-expressed in the muscle cells of these worms to serve as a transformation marker. We then performed dual whole-cell voltage clamp recordings on two contiguous neighboring ventral muscle cells from two different rows of muscle cells within the same quadrant (jI) were measured from one muscle cell held constant at –30 mV while a series of membrane voltage steps  were applied to the other neighboring muscle cell of the pair from a holding voltage of –30 mV  relationships and quantified the junctional conductance (jG) from the slope of the linear portion of the j - VjI curves. While jI was prominent in the wild type, unc-9(e101) mutant showed very little jI (unc-9(fc16) null mutants analyzed in our earlier studies (unc-9(e101) mutants, while unc-9(e101) mutants expressing unc-9(ΔN18) completely lacked the intra-quadrant coupling similar to unc-9(e101) mutants  can rescue the electrical coupling defect of quadrant . Junctiof –30 mV . We plotittle Ij , similar studies . As expe mutants . These runc-9(ΔN18) transgenes could be due to the nature of overexpression of the transgene from extra-chromosomal concatemer arrays. To exclude this possibility, we generated unc-9(ΔN18) mutant using CRISPR/Cas9 genome editing. Similar to unc-9(e101) mutant, unc-9(syb3236 [unc-9(ΔN18)]) mutant animals exhibit a kinker phenotype (unc-9(syb3236 [unc-9(ΔN18)]) mutant did not exhibit a presynaptic tiling defect between DD5 and DD6 in the egl-20(n585) mutant background (unc-9(ΔN18) deletion did not cause a presynaptic tiling defect in the egl-20(n585); unc-1(miz297) null mutant background where the UNC-9 gap junction channel activity is absent . We first tested whether UNC-9::mTagBFP2 forms an open gap junction channel similar to UNC-9::GFP by testing whether it can rescue the electrical coupling defects of unc-9(e101) mutant. The muscle expression of unc-9::mTagBFP2 in unc-9(e101) mutant resulted in a much larger jI and jG than those obtained with wild type unc-9 , pan-neuronal expression of unc-9::mTagBFP2 rescued the presynaptic patterning defect of egl-20(n585); unc-9(e101) mutant (unc-9::mTagBFP2 did not rescue the kinker phenotype of unc-9(e101) mutant (egl-20(n585); unc-9(miz81 [unc-9::7×gfp11]) mutant. UNC-9::7×GFP11, which by itself is not fluorescent, has seven tandem repeats of GFP11 at the carboxy-terminus of UNC-9, and hence, is expected to have a similar effect on gap junction function as UNC-9::GFP or UNC-9::mTagBFP2. While UNC-9::7×GFP localizes to the putative gap junction sites between DD5 and DD6 axons, unc-9(miz81 [unc-9::7×gfp11]) mutants exhibit a kinker phenotype (egl-20(n585); unc-9(miz81 [unc-9::7×gfp11]) mutant does not exhibit the presynaptic tiling defect  mutant . Importa) mutant , which ihenotype , suggestg defect . These rC. elegans. We found that EGL-20/Wnt negatively regulates the length of the posterior axon and dendrite of DD5. We showed that presynaptic tiling requires UNC-9/INX, while the position of postsynaptic spines appears to depend on the length of the posterior dendrite. UNC-9 is localized at the presynaptic tiling border between DD5 and DD6 where it forms gap junction channels. Strikingly, the gap junction channel activity of UNC-9 is dispensable for its function in establishing presynaptic tiling.Here we established a novel system to study neuronal and synaptic tiling in the DD type GABAergic motor neurons in Drosophila where axonal tiling defects cause disrupted synaptic connections ; unc-9(e101) mutant is smaller than the degree of axonal tiling defect, suggesting the presence of additional factors that regulate presynaptic patterning between DD5 and DD6 along with unc-9. Previously, we showed that spatial expression pattern of two Wnt proteins (LIN-44 and EGL-20) defines topographic presynaptic patterning of DA8 and DA9 in the absence of Plexin-dependent presynaptic tiling mechanism  background ; unc-9(e101) double mutants exhibit presynaptic tiling defects  cholinergic motor neurons in C. elegans , nor the upstream components that are required for UNC-9 localization at the presynaptic tiling border. There are several genes that are necessary for the proper clustering of gap junction channels. However, UNC-9 localization was unaffected in the mutants of  elegans . We obsedbn-1(ok925), egl-20(n585), inx-1(miz280), inx-2(miz282), inx-10(miz284), inx-14(ok3267), mig-15(rh148), nlg-1(yv15), nlr-1(miz202), pes-7(gk123), plx-1(nc36), rap-1(miz203), rap-2(gk11), unc-1(e719), unc-1(miz297), unc-7(e5), unc-9(e101), unc-9(tm5479), unc-9(miz81 [unc-9::7×gfp11]), unc-9(syb3236 [unc-9ΔN18]), unc-24(miz225), unc-24(miz226), unc-104(e1265), nlr-1(gk366849), vab-9(ju6), zoo-1(tm4133). Genotyping primers are listed in the supplemental material.Bristol N2 strain was used as wild type reference. All strains were cultured in the nematode growth medium with OP50 as described previously  at 25°C.The transgenic lines with extrachromosomal arrays were generated using the standard microinjection method . The intmizIs3 ; wyIs292 ::nlg-1::yfp); wyIs442 ; wyIs486 ; juIs463 ; ufIs126 (Pflp-13::acr-12::GFP); mizEx69, mizEx407 ; mizEx72, mizEx420 (Prgef-1::unc-9(ΔN18), Podr-1::GFP); mizEx370, mizEx371 ; mizEx416 ; mizEx429, mizEx433 (Pflp-13::unc-9(ΔN18), Podr-1::GFP); mizEx430 ; mizEx510 , mizEx496, mizEx497 ; mizEx499, mizEx500 (Pmyo-3::unc-9(ΔN18), Pmyo-3::GFPnovo2, Podr-1::GFP); mizEx498, mizEx499 (Pmyo-3::unc-9(ΔN18), Pmyo-3::GFPnovo2, Podr-1::GFP); mizEx500, mizEx501 ; mizEx515 ; mizEx543, mizEx544 ; mizEx546, mizEx547 ; mizEx548, mizEx549 ; mizEx550, mizEx551 ; Podr-1::GFP); mizEx558, mizEx559 .The following transgenes were used in this study: egl-20(n585); wyIs442UJ1044 egl-20(n585); unc-9(e101); wyIs442UJ1215 egl-20(n585); unc-9(syb3236 [unc-9ΔN18]); wyIs442UJ1543 unc-9(miz81 [unc-9::7×gfp11]); juIs463UJ1261 C. elegans expression clones were made in a derivative of pPD49.26 (A. Fire), the pSM vector (a kind gift from S. McCarroll and C. I. Bargmann). unc-9 cDNAs were amplified with Phusion DNA polymerase (NEB) from N2 cDNA library synthesized with superscript III first-strand synthesis system (Thermo Fisher Scientific). The amplified cDNAs were cloned into the AscI and KpnI sites of pSM vector using Gibson assembly method . The 7×gfp11 sequence was cloned into the SacII site of the loxP::myo-2::NeoR plasmid using Gibson assembly  strain (unc-9#1:AATTAAACCCCATTTCAGGA.pTK73 plasmid was used as a backbone vector for gRNA expression plasmid construction for ) strain . The folunc-9(miz81 [unc-9::7×gfp11]), the unc-9 repair template plasmid, unc-9 sgRNA plasmid, and Cas9 plasmid . Progenies without pharyngeal Pmyo-2::GFP expression were isolated. Successful genome-edited candidate animals were confirmed via PCR and Sanger sequencing.To generate  plasmid  were co- plasmid . F1 proginx-1(miz280), inx-2(miz282), inx-10(miz284), nlr-1miz202 rap-1(miz203), unc-1(miz297), and unc-24(miz225) & (miz226) mutants, we used a direct injection of gRNA/Cas9 complex with the homology-dependent repair (HDR) donor oligos (unc-24(miz225) and (miz226) alleles have an identical mutation.To generate r oligos . unc-24: Gly222Stopinx-2(miz282): Gly138Stopinx-10(miz284): Phe138Stopnlr-1(miz202): Asn325Stoprap-1(miz203): Ser17Stopunc-1(miz287): Isoform A-Ser128Stop;Isoform B-Ser132Stopunc-24(miz225) & (miz226): Ala281Stopinx-1GGTACTGACAACCTTTTTTAgRNA: HDR donor oligos:CAAGTGTTTATGTTAAACAGTTTCCTTGGTACTGACAACCTTTTTTACtgagaattcTTCACATTTTGAGAGATTTGTTGAATGGTCGTGAATGForward:CATTCACGACCATTCAACAAATCTCTCAAAATGTGAAgaattctcaGTAAAAAAGGTTGTCAGTACCAAGGAAACTGTTTAACATAAACACTTGReverse:inx-2AGAACCACAATTAACCTCAAgRNA: HDR donor oligos:CATCTCTGGAATTTGTTTCACAAAAGAACCACAATTAACCTCAAAtgagaattcCTTTAAGATTTTTTGAAGGAGCTTTGAAAAAATTGGAGForward:CTCCAATTTTTTCAAAGCTCCTTCAAAAAATCTTAAAGgaattctcaTTTGAGGTTAATTGTGGTTCTTTTGTGAAACAAATTCCAGAGATGReverse:inx-10ACGGTAGATTGAAAAGCCAAgRNA: HDR donor oligos:CATCGACGCCTGCAAAAGAAACAAATTCGTCCGCATCGTTgagaattcTTGGCTTTTCAATCTACCGTACTCTGCATTCTTTGTGACTGCAATGForward:CATTGCAGTCACAAAGAATGCAGAGTACGGTAGATTGAAAAGCCAAgaattctcAACGATGCGGACGAATTTGTTTCTTTTGCAGGCGTCGATGReverse:nlr-1GTTACAAGTGGACCATTGATgRNA: HDR donor oligos:CATTCACTTTCCTTCCGTTTTCATTCGAGTTCCGGATAGTTgaattcTAATCAATGGTCCACTTGTAACACTTTTTGATGCTGAAAATGGAACForward:GTTCCATTTTCAGCATCAAAAAGTGTTACAAGTGGACCATTGATTAgaattcAACTATCCGGAACTCGAATGAAAACGGAAGGAAAGTGAATGReverse:rap-1AAGATTGTTGTGCTCGGATCgRNA: HDR donor oligos:caaaaaaagaATGCGGGAGTATAAGATTGTTGTGCTCGGATCTGaggatccgGAGGAGTAGGAAAATCCGCACTGgtatagatgagagtgaccggForward:ccggtcactctcatctatacCAGTGCGGATTTTCCTACTCCTCcggatcctCAGATCCGAGCACAACAATCTTATACTCCCGCATtctttttttgReverse:unc-1ACCCGATTGCTTCCGTCAACgRNA: HDR donor oligos:GTATCCGTCGACGCCGTTGTCTACTTCCGTACCTgagaattcACCCGATTGCTTCCGTCAACAATGTTGACGATGCCATTTACTCCACCAAACTGCForward:GCAGTTTGGTGGAGTAAATGGCATCGTCAACATTGTTGACGGAAGCAATCGGGTgaattctcAGGTACGGAAGTAGACAACGGCGTCGACGGATACReverse:unc-24CGTTGAGCAGCGTTGCGAAAgRNA: HDR donor oligos:GGAAGGAGAGAACATGGGGATGTCTGCGTTGAGCAGCGTTGCGAAAgaattcTGATGCTGGTCAACAGTTGTGGCAAGTTATTGGACCAGTATTCGForward:CGAATACTGGTCCAATAACTTGCCACAACTGTTGACCAGCATCAgaattcTTTCGCAACGCTGCTCAACGCAGACATCCCCATGTTCTCTCCTTCCReverse:C. elegans using a Zeiss LSM800 Airyscan confocal microscope  with oil immersion lens 40× and 63× magnification . Worms were immobilized on 5% agarose pads using a 3:1 mixture of 0.225 M 2,3-butanedione monoxime (Sigma-Aldrich) and 7.5 mM levamisole (Sigma-Aldrich). Images were analyzed using Zen software (Carl Zeiss). Images were straightened with ImageJ . A range of 18–24 Z-stack images were taken for each animal to encompass the cell bodies, axons, and synapses of the DD5 and DD6 neurons. L4.4–L4.6 larval stage animals, judged by the stereotyped shape of the developing vulva  were captured in live ng vulva , were usStereoscope images were taken on Zeiss Stemi 305 with Zeiss Labscope.For quantifying fluorescent signal intensity of UNC-9::7×GFP, 21 Z-stacks are compiled using ImageJ (Image → Stacks → Z-project → Sum Slices). The signal intensity of 7.33 µm of region of interest (ROI) at the anterior tip of the DD6 axon is measured using ImageJ . The signal from the region adjacent to the ROI is used as background signal which was subtracted from the signal intensity of UNC-9::7×GFP.Generated data were analyzed and processed by Prism9 . We applied the one-way ANOVA method with post hoc Tukey’s multiple comparison test  of both cells was held at –30 mV, from which a series of voltage steps  were applied to one cell (Cell 1), whereas the other cell (Cell 2) was held constant to record j. VjI was defined as mV of Cell 2 minus mV of Cell 1. Series resistance was compensated to approximately 80% in the voltage-clamp experiments. Data was sampled at a rate of 10 kHz after filtering at 2 kHz. The bath solution and pipette solution used were identical to those described earlier (Adult (day 1) hermaphrodite animals were immobilized and dissected as described previously . Briefly earlier .dbn-1(ok925)CTGCGCTTATTATCGTCCTG 3’Forward: 5’ CGATCAAGTCGATAGCGGATG 3’Wild type reverse: 5’ GCACGGCCGCGGCAATTCACAC 3’Mutant reverse: 5’ egl-20(n585) – wild type PCR product will be digested by HpyCH4VCTCTTAAAAACTTACCTCTCAAATTTGAACTTATTCTTGC 3’Forward: 5’ CCTCATTACCATTCAACTGATAG 3’Reverse: 5’ inx-1(miz280) – mutant PCR product will be digested by EcoRICAATGTGCAATCATTGACTCAAATGGC 3’Forward: 5’ CATCATACAGTCCAATGCGTGCTAATG 3’Reverse: 5’ inx-2(miz282) – mutant PCR product will be digested by EcoRIGTATGAGCAACTAGTAGGGATGCTGCG 3’Forward: 5’ CCTAACTTGAGCAAGAAGTAATTGATGGTAGCTTG 3’Reverse: 5’ inx-10(miz284) – mutant PCR product will be digested by EcoRIGAGATTGTCAAGTTATCTTCGGATCCC 3’Forward: 5’ CCAGCACACATTGTATAGTATGTTC 3’Reverse: 5’ nlr-1(miz202) – mutant PCR product will be digested by EcoRIGTTGTAAGGTCAACTATTAGAGCAAGG 3’Forward: 5’ GTTCGAGTTGATGGAAACGTGACAAG 3’Reverse: 5’ nlr-1(gk366849) – wild type PCR product will be digested by MfeIGTTTGCTCTCTTCATCAATCACTACATCC 3’Forward: 5’ CGCCATAAAACGATATATTATGTGTAG 3’Reverse: 5’ pes-7(gk123)GTGTAGGTGTGAGGAAGTCC 3’Forward: 5’ CGGCGTCGCATCATTCTGCCG 3’Wild type reverse: 5’ CCGCCAATTAGATTTGTTCG 3’Mutant reverse: 5’ plx-1(nc36)CTTCGAGAGCCCCCTCATTCTTAATG 3’Forward: 5’ CCGGCACACGTTAAACTAGTGCTACCG 3’Reverse 5’ rap-1(miz203) – mutant PCR product will be digested by BamHIGTGTCATCTGGTCTGTACTTGGTGC 3’Forward: 5’ GGAGTACTGTAACTAAAAACTCTCGTC 3’Reverse: 5’ rap-2(gk11)TCTCATCTCCATCGTCGTTCCTGC 3’Forward: 5’ TCCATTCACTGAATGTTCCGC 3’Wild type reverse: 5’ GAGGGAGTTCAAAGTGGTCGTTC 3’Mutant reverse: 5’ unc-1(miz297) – mutant PCR product will be digested by EcoRICTGACCTGGGCTCACTTTTAAGTC 3’Forward: 5’ GCCCCAAATATGAGCATTGAGTGATCAG 3’Reverse: 5’ unc-24(miz225) & (miz226) – mutant PCR product will be digested by EcoRICCACAGA TCGTGGTCTCGTGGAAC 3’Forward: 5’ CTGACATTCGCTCCACCAAGTGTTTTAGC 3’Reverse: 5’ zoo-1(tm4133)CAGGTCGGCGGAAGTGTCGGAGTACGTG 3’Mutant forward: 5’ CCGAATCAAGCGACCGCCGAGCAAATTGC 3’Wild type forward: 5’ GTGCCAGCTGAAGACGTTCAACAGACTCG 3’Reverse: 5’  This work provides a highly valued addition to our understanding of innexin gene function in the nervous system. The authors describe here potential functions in synapse tiling. The paper should be of interest to researchers with an interest in molecular mechanisms governing nervous system development. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:C. elegans" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Piali Sengupta as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Michael M Francis (Reviewer #3).Thank you for submitting your article "Channel-independent function of UNC-9/INX in spatial arrangement of GABAergic synapses in The reviewers have discussed their reviews with one another.As you will see in the reviews below, all three reviewers thought that this paper has very good potential, but that a number of revisions are required to make the paper become potentially acceptable. Each one of the 3 reviewers has non-overlapping sets of suggestions and we all felt that in aggregate these revisions are all doable .Reviewer #1 (Recommendations for the authors):This reviewer suggests using heat shock promoter to express UNC-9::GFP to check whether late expression of UNC-9 could revert the presynaptic pattering defect observed in egl-20; unc-9 double mutant animals.Reviewer #2 (Recommendations for the authors):A few questions need to be addressed for current version of the manuscript.1) Confirm UNC-9 localization using anti-UNC-9 antibody. As evidenced in this study UNC-9::GFP is a gain-of-function version of UNC-9, and it may or may not represent UNC-9 localization in vivo.2) Test whether unc-9 is also required for DD5/DD6 synapse tilling in other genetic backgrounds, in which DD5 and DD6 have overlapping axons. one concern is whether unc-9 is only required for synapse tilling when egl-20 is mutated.3) DD5/DD6 don't have overlapping axons in wild type animals, and therefore all experiments for DD5/DD6 synapse tilling can only be carried out in mutants with abnormally overlapped DD5/DD6 axons. One concern is how "synapse tilling" in these abnormally developed axons represents synapse tilling during normal development. I understand it is challenging to address this in DD neurons, but it should be testable in other neurons who have overlapping axons and tilled synapses, for example DA neurons.Reviewer #3 (Recommendations for the authors):My major comment relates to the potential advance offered by the studies. The impact of the findings would be enhanced if the authors were able to define mechanisms for UNC-9 localization and/or function in tiling. Nonetheless, the findings of the study as presented are provocative and suggest a potentially novel mechanism for synaptic tiling through regulated expression/localization of gap junction proteins. This will be of interest to the field and sets the stage for follow up studies of mechanism.In several instances, the writing is unclear:Line 148: "we observed minimal overlaps between the axons and the dendrites of DD5 and DD6" would be more clearly stated as "minimal overlap between either axons or dendrites of…"Lines 168-9 are redundant with lines 173-4Lines 206-7: "the position of DD5 postsynaptic dendritic spine is determined by the length of DD5 dendrite". This is unclear, ACR-12::GFP puncta appear to extend along the full length of the dendrite.Lines 275-6: Delete "both". Reviewer #1 (Recommendations for the authors):This reviewer suggests using heat shock promoter to express UNC-9::GFP to check whether late expression of UNC-9 could revert the presynaptic pattering defect observed in egl-20; unc-9 double mutant animals.We have conducted this experiment using a heat-shock promoter.Reviewer #2 (Recommendations for the authors):A few questions need to be addressed for current version of the manuscript.1) Confirm UNC-9 localization using anti-UNC-9 antibody. As evidenced in this study UNC-9::GFP is a gain-of-function version of UNC-9, and it may or may not represent UNC-9 localization in vivo.We thank the reviewer for their comment. Due to the strong expression of UNC-9 in the body wall muscle cells, we do not think we could reliably visualize endogenous UNC-9 gap junction formed specifically between DD neurons. However, we believe that UNC-9::7×GFP represents the endogenous UNC-9 localization for the following reasons. First, in our previous work, we showed that UNC-9::GFP exhibits the same localization pattern as endogenous UNC-9 detected with the anti-UNC-9 antibody in the body wall muscles . Second, in the revised manuscript, we examined the localization of N-terminally tagged GFP::UNC-9 which was shown to be functional . We observed that GFP::UNC-9 was localized at the axonal and dendritic tiling border of DD5 and DD6 similar to UNC-9::7×GFP. We therefore believe that UNC-9::7×GFP can be used to visualize the subcellular localization of UNC-9 even though it has a compromised channel activity. We have included the representative images of GFP::UNC-9 in Figure 2H.2) Test whether unc-9 is also required for DD5/DD6 synapse tilling in other genetic backgrounds, in which DD5 and DD6 have overlapping axons. one concern is whether unc-9 is only required for synapse tilling when egl-20 is mutated.sax-2(ky216) mutant exhibits a mild axonal tiling defect (see sax-2(ky216); unc-9(e101) double mutants and found that there is a tendency that unc-9(e101) enhances the presynaptic tiling defect in sax-2(ky216) mutants, despite no statistically significant differences. This is likely due to the smaller degree of axonal tiling defect in sax-2(ky216) mutant compared with egl-20(n585) mutant, which limits the axonal region to form ectopic synapses compared with egl-20(n585) mutant.From a candidate screening, we found that the fect see . We exam3) DD5/DD6 don't have overlapping axons in wild type animals, and therefore all experiments for DD5/DD6 synapse tilling can only be carried out in mutants with abnormally overlapped DD5/DD6 axons. One concern is how "synapse tilling" in these abnormally developed axons represents synapse tilling during normal development. I understand it is challenging to address this in DD neurons, but it should be testable in other neurons who have overlapping axons and tilled synapses, for example DA neurons.unc-9(e101) mutant animals and did not observe presynaptic patterning defect. In DA9, UNC-9::7×GFP is predominantly localized at the distal dendrite. In the axon, we observed UNC-9::7×GFP puncta throughout the axon but we did not observe consistent localization in relation to their presynaptic domain .We examined presynaptic tiling between DA8 and DA9 in the Reviewer #3 (Recommendations for the authors):My major comment relates to the potential advance offered by the studies. The impact of the findings would be enhanced if the authors were able to define mechanisms for UNC-9 localization and/or function in tiling. Nonetheless, the findings of the study as presented are provocative and suggest a potentially novel mechanism for synaptic tiling through regulated expression/localization of gap junction proteins. This will be of interest to the field and sets the stage for follow up studies of mechanism.In several instances, the writing is unclear:Line 148: "we observed minimal overlaps between the axons and the dendrites of DD5 and DD6" would be more clearly stated as "minimal overlap between either axons or dendrites of…"Lines 168-9 are redundant with lines 173-4Lines 206-7: "the position of DD5 postsynaptic dendritic spine is determined by the length of DD5 dendrite". This is unclear, ACR-12::GFP puncta appear to extend along the full length of the dendrite.Lines 275-6: Delete "both".We thank the reviewer for pointing these out, we have rectified the errors including the ones indicated above.