Multiple processes exist in a cell to ensure continuous production of essential proteins either through cap-dependent or cap-independent translation processes. Viruses depend on the host translation machinery for viral protein synthesis. Therefore, viruses have evolved clever strategies to use the host translation machinery. Earlier studies have shown that genotype 1 hepatitis E virus (g1-HEV) uses both cap-dependent and cap-independent translation machineries for its translation and proliferation. Cap-independent translation in g1-HEV is driven by an 87-nucleotide-long RNA element that acts as a noncanonical, internal ribosome entry site-like (IRESl) element. Here, we have identified the RNA-protein interactome of the HEV IRESl element and characterized the functional significance of some of its components. Our study identifies the association of HEV IRESl with several host ribosomal proteins, demonstrates indispensable roles of ribosomal protein RPL5 and DHX9 (RNA helicase A) in mediating HEV IRESl activity, and establishes the latter as a bona fide internal translation initiation site.IMPORTANCE Protein synthesis is a fundamental process for survival and proliferation of all living organisms. The majority of cellular proteins are produced through cap-dependent translation. Cells also use a variety of cap-independent translation processes to synthesize essential proteins during stress. Viruses depend on the host cell translation machinery to synthesize their own proteins. Hepatitis E virus (HEV) is a major cause of hepatitis worldwide and has a capped positive-strand RNA genome. Viral nonstructural and structural proteins are synthesized through a cap-dependent translation process. An earlier study from our laboratory reported the presence of a fourth open reading frame (ORF) in genotype 1 HEV, which produces the ORF4 protein using a cap-independent internal ribosome entry site-like (IRESl) element. In the current study, we identified the host proteins that associate with the HEV-IRESl RNA and generated the RNA-protein interactome. Through a variety of experimental approaches, our data prove that HEV-IRESl is a bona fide internal translation initiation site. Many cellular and viral mRNAs are translated by cap-independent mechanisms by using distinct cis-acting regulatory elements, such as cap-independent translational enhancers (CITEs), N6-methyladenine (m6A) modification of the mRNAs, or internal ribosome entry sites (IRESs). Initiation of cap-dependent translation occurs through a ribosomal scanning mechanism in which the 48S initiation complex moves along the mRNA from the m7G cap in the 5′ to 3′ direction to locate a suitable AUG initiation codon. Following AUG recognition, through a series of steps, the 80S ribosome complex is assembled on the mRNA with initiator methionyl-tRNA (met-tRNAi) bound to the AUG codon at the P-site, leading to translation elongation N manner 68.pro cleaves the eIF4E binding N-terminal domain of eIF4G without affecting its eIF3/eIF4A binding property. Stable association of eIF4G with PV IRES domain V promotes binding of other initiation factors, leading to formation of the 43S preinitiation complex. The FMDV IRES is a well-studied example of a type II IRES. Domain IV of the FMDV IRES binds the scaffold protein eIF4G. The 3Cpro and Lpro of FMDV cleave eIF4G similar to 2Apro of type I IRESs. Notably, the FMDV IRES skips ribosomal scanning; instead, the IRES-proximal stem-loop formation brings 84 nucleotides (nt) downstream of AUG close to the first AUG to start translation by direct ribosome transfer. Other identified cis-acting elements for type II IRES activity are the GNRA, RAAA, and C-rich loops in domain III /HCV-like IRES. The 5′ UTR of HCV contains four domains; domains I and II are involved in viral replication, while domains III and IV are involved in IRES activity  and foot and mouth disease virus (FMDV), respectively. The PV IRES harbors six stem-loops (SLs) designated domains I to VI. Domain I forms a unique clover leaf structure and is critical for replication of both positive- and negative-sense RNA. Domains II to VI are responsible for PV IRES activity. During PV infection, viral 2Amain III . The typmain III  and requmain III . Type IVactivity , 13. Thee family , 13. IGRe family , 15.Many viruses have evolved the ability to use both cap-dependent and cap-independent translation processes depending on the state of the cell. Notably, although dengue virus and Zika virus genomic RNAs are usually translated through a cap-dependent mechanism, their 5′ UTRs also contain IRES elements, which maintain viral translation under unfavorable conditions where cap-dependent translation is inhibited by the host –18. Simi–19–Hepeviridae. The viral genome is capped at the 5′ end followed by a short UTR of 25 nucleotides. The viral nonstructural proteins are synthesized from open reading frame 1 (ORF1) located between nucleotides 26 and 5107. Viral capsid protein is produced by ORF2 located at the 3′ end between nucleotides 5145 and 7127. A short 3′ UTR of 65 nucleotides is present downstream of ORF2, followed by a poly(A) tail (–Hepatitis E virus (HEV) is a positive-strand RNA virus of the family (A) tail –30. The A) tail –.in vitro translation assays or bicistronic reporter-based assays. Analysis of cap-independent translation driven by the HEV RNA regulatory element by bicistronic reporter assays along with site-directed mutagenesis-mediated mapping of the regulatory RNA sequence suggested that the HEV RNA regulatory element is an IRES element. However, considering the small size of the regulatory element and lack of strong homology with canonical IRES elements, it was designated an IRESl element. The current study was designed to further explore the mechanism of HEV IRESl activity and verify its function as a bona fide internal translation initiation site. Host interaction partners of HEV IRESl were identified, and their importance in IRESl-mediated translation was evaluated. Subsequent experiments showed the association of HEV IRESl RNA with actively translating ribosomes, supporting its role as a translation initiation element. The significance of these findings in validating the role of HEV IRESl as a bona fide translation initiation site is discussed.Our earlier study demonstrated the coexistence of both cap-dependent and cap-independent translation in g1-HEV . AlthougAF444002.1) corresponding to the IRESl region were cloned into the pRMB vector between the BirA ligase-binding stem-loop (RMB-SLI and RMB-SLII) sequences  were used to identify host proteins that interact with HEV IRESl RNA. The RaPID assay involves biotinylation of IRES-interacting proteins, followed by their identification by liquid chromatography with tandem mass spectroscopy LC-MS/MS) . The RaPequences  and C. A-MS/MS . equences . An unreThe quality of the MS data between replicates of different samples was checked by Pearson correlation analysis, which showed good correlation (average range of 0.2 to 1.0). Proteins with at least one biotinylated peptide with a posterior error probability (PEP) score greater than or equal to the median value in the Gaussian smoothing curve of each sample were selected for further analysis. Specific interaction partners of HEV IRESl RNA were selected by subtracting the biotinylated HEV IRESl data set from the biotinylated HEV (618 to 738), unbiotinylated HEV (618 to 738), and unbiotinylated HEV IRESl data sets ; Table 1lacZ and HIS3 reporter genes by HEV IRESl RNA was checked by cotransforming the plasmids encoding HEV IRESl (p3HR2-HEV IRESl) and the GAL4 activation domain (pACT2) into chemically competent YBZ1 yeast cells, followed by selection of the cotransformants on medium lacking leucine, uracil, and histidine (LUH−) and assessment of β-galactosidase activity. No growth of colonies was seen on LUH− plates nor was β-galactosidase activity observed, indicating that there was no self-activation of the reporter genes by the bait RNA . Note that 3-AT is a competitive inhibitor of the histidine biosynthesis pathway. Therefore, addition of 3-AT allows growth of the cotransformants that show strong interaction between the bait RNA and the prey protein. Three hundred and ninety-five colonies were obtained in the primary screening, of which the human liver cDNA insert could be detected in 285 colonies (Table S2). Restriction pattern analysis of the prey plasmid DNA isolated from the 285 colonies showed that the unique cDNA insert was present in 75 plasmids. Analysis of the cDNA insert sequences identified 8 protein-coding genes, which were in frame with the GAL4 activation domain (Table S2). Retransformation of those 8 plasmids along with the p3HR2-HEV IRESl plasmid showed that all of them were able to interact with the HEV IRESl , 3635, 3bait RNA . Next, tEV IRESl . The eigEV IRESl .−) and (ii) by measuring the interaction of those proteins with the known IRES sequences from the genotype 3A hepatitis C virus (HCV) and the foot and mouth disease virus (FMDV). All sequences were cloned into the p3HR2 vector upstream of the MS2 coat protein-binding stem-loop RNA sequences . No significant interaction was observed between HEV IRESl− RNA and the host proteins  by measuring the interaction of those proteins with an RNA sequence complementary to the HEV IRESl (HEV IRESlproteins . Y3H anain vitro pulldown assay using in vitro-transcribed biotinylated HEV IRESl RNA as bait. Nonbiotinylated HEV IRESl and biotinylated control RNA (nucleotides 618 to 738 in the g1-HEV genome) as well as cell lysate only (mock) were used as controls to ensure specificity of the assay  network, as described earlier . Analysi × 10−16 . The aboNext, Gene ontology (GO) and Reactome pathway analysis of the HEV IRESl RPPI network was performed using the “Enrichr” tool to determine the significantly enriched processes/pathways. Proteins involved in translation and mRNA catabolic processes were enriched in the GO biological processes category, supporting involvement of the HEV IRESl element in protein synthesis . In addi37–In silico molecular docking has been used to determine the complex structures of RNA-protein interactions  of hepatitis A virus . RPL5, RPL24, and DHX9 siRNAs partially reduced global translation, while RPL26, RPL41, and RPS7 siRNAs increased translation  against some of the host factors to deplete them in Huh7 cells, followed by measurement of HEV IRESl activity and g1-HEV replication. Treatment of Huh7 cells for 72 h with siRNAs against RPL5, RPL24, RPL26, RPL41, RPS3A, RPS7, and DHX9 depleted the corresponding proteins . Note thnslation , top. A nslation , bottom.Renilla luciferase [Renilla-Luc] activity) in siRNA-treated Huh7 cells, as described earlier (Renilla-Luc (cap-dependent translation activity)  was used to simultaneously measure HEV IRESl activity (by measuring firefly luciferase [Firefly-Luc] activity) and cap-dependent translation activity   and C. Lilla-Luc  and C. Willa-Luc . DHX9 siilla-Luc . These dNext, g1-HEV-expressing Huh7 cells were treated with different siRNAs, followed by measurement of the level of viral sense-strand RNA. Lack of RPL5 and DHX9 proteins significantly reduced the level of g1-HEV RNA . HoweverRenilla-Luc reporter driven by cap-dependent translation was used as a positive control for the assay (pRL-TK). A control RNA (pGL3 control RNA) was used to check specificity of the polysome fractions. HEK293T cells were cotransfected with the pSuper-HEV IRESl and pRL-TK plasmids. Forty-eight hours posttransfection, cells were lysed, and Firefly-Luc and Renilla-Luc activity was measured. As expected, significant activity of both Firefly-Luc and Renilla-Luc was observed . As expected, Renilla-Luc RNA was detected in the anti-m7G cap antibody-immunoprecipitated sample, whereas Firefly-Luc RNA was not detectable in aliquots of the same sample  or puromycin (Puro), which stalls translating ribosomes or prevents polysome assembly, respectively  sucrose density gradient centrifugation, followed by measurement of both Firefly-Luc and ectively , 45. Ribolysomes . On the olysomes . Next, aolysomes . DHX9 waolysomes . PPIG waolysomes . PPIG wa proline . CollectRenilla-Luc-expressing HEK293T cells treated with different siRNAs and cycloheximide. Fractions 1, 5, 9, and 13 from the density gradient centrifugation samples were analyzed to quantify the levels of Firefly-Luc and Renilla-Luc RNAs as free RNA (fraction 1), in the 40S-60S ribosome complex (fraction 5), or in the polysome complex (fractions 9 and 13). Treatment of cells with siRNAs against RPL5, RPL26, and DHX9 significantly reduced HEV IRESl RNA association with polysomes, whereas siRNAs against RPL24, RPL41, and RPS7 had no effect  in polysomes.The importance of HEV IRESl RNA-binding translation-related proteins in driving the association between HEV IRESl RNA and polysomes was further assessed by a ribosome fractionation assay in Firefly-Luc- and o effect . Howeverolysomes . Next, aolysomes . CollectWhile investigating the cause of poor replication of g1-HEV in Huh7 cells, we observed better viral replication after treatment of cells with ER stress-inducing compounds such as tunicamycin and thapsigargin. Subsequently, we identified a fourth ORF in the viral genome, which produces the ORF4 protein that plays a key role in assembly of the viral replication complex . We alsoTwo independent approaches were used to identify the host interaction partners of HEV IRESl RNA: the RaPID assay and the Y3H assay. The RaPID assay identifies stable and transient interactions of all proteins associated with the RNA of interest that include both direct and indirect interaction partners of the RNA. Several controls were used to avoid/subtract nonspecific interaction partners, thus ensuring the generation of high-confidence mass spectrometry data for downstream studies. For example, the good quality of our HEV IRESl RaPID assay LC-MS/MS data is evident from the fact that comparison of the HEV IRESl-interacting proteins with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 5′- and 3′-UTR RNA-interacting proteins (reported by our laboratory using the same technique) identified only RPL7a as a common protein . RPL7a iA limitation of the RaPID assay is attributed to its dependence on biotinylation of lysines exposed on the surface of RNA-binding proteins present within a distance of 10 nm from the BirA ligase. Thus, it is possible to lose some of the RNA-binding proteins if they are buried deep inside the complex and are inaccessible to BirA ligase for biotinylation. Therefore, a conventional Y3H assay-based human liver cDNA library screening was conducted in parallel to identify the interaction partners of HEV IRESl RNA. The Y3H assay offers the advantage of identifying the direct interaction partners of a bait RNA and identified ribosomal proteins , a known DNA-binding protein (GREBP), and a cyclophilin family protein (PPIG) as interaction partners of HEV IRESl , 49. Spein vitro biotinylated RNA pulldown assay and molecular docking analysis were performed for some of the HEV IRESl-interacting proteins, which supported their interaction. Docking analysis also demonstrated that the complex between HEV IRESl and RPL5 was more stable than the others. Moreover, comparative interaction mapping showed that the interaction between the HEV IRESl element and the ribosomal proteins primarily involved SL A , SL B , and the unpaired region between SL B and SL C  of the former .G) for interaction with HEV IRESl RNA in silico. RPL5 is also a component of the polysome, and lack of RPL5 significantly affected polysome stability and dissociated both HEV IRESl and Renilla-Luc RNAs from the polysome. Hence, it is clear that RPL5 is an essential factor for both cap-dependent and cap-independent translation. RPL26 showed a pattern similar to RPL5 by inhibiting HEV IRESl-Luc and Renilla-Luc activities. Moreover, the HEV IRESl Firefly-Luc and Renilla-Luc RNA association with polysomes was significantly reduced after treatment of cells with RPL26 siRNA. However, RPL26 differed from RPL5 in the following parameters: it bound moderately with HEV IRESl RNA both in Y3H assays and in in silico analyses, a lack of RPL26 did not affect g1-HEV replication, and a lack of RPL26 did not affect polysome stability. Earlier studies have shown that the lack of RPL26 does not affect cap-dependent translation in Saccharomyces cerevisiae, and RPL26 is a known IRES transacting factor (ITAF), which has been shown to control DNA damage-induced cap-independent translation of p53 mRNA -mediated silencing of CDK6 , HNRNPA2/B1, YTHDF2, EEF2, and LARP4 were also identified as interaction partners of HEV IRESl RNA. RHA is known to associate with the IRESs of FMDV and HCV and acts as an ITAF, promotes translation from the IRES of retroviral RNA and ue virus –59. RHA e virus –. RHA is e virus –, 62. In  mitosis . The mecechanism . It shou of CDK6 . An earl of CDK6 . YTHDF2  of CDK6 , 67. Euk of CDK6 . LARP4 b of CDK6 –71. The XBP1 RNA and prevents its degradation  and the BASU RaPID plasmid  were gifted by Paul A. Khavari  and were reported earlier (AF444002.1) were PCR amplified from the pSK HEV2 plasmid (GCTTGGTGCCGATCGGTCCC-3′) and IRESl87 BASU CBB RP (5′-AGCTCATCTGGCAGCAAGCTCAG-3′) and were ligated with the pRMB vector backbone generated by BSMB1 restriction enzyme digestion, followed by blunting. For hybrid RNA generation for the Y3H assay, HEV IRESl, FMDV IRES, and HCV IRES sequences were PCR amplified from the pSKHEV2 plasmid, pVitro2-neo-mcs plasmid , and S52/SG-Feo(AI) replicon (gifted by Charles M. Rice  acetonitrile and 0.1% [vol/vol] formic acid in water) for LC-MS/MS analysis.(iii) LC-MS/MS acquisition. A Sciex 5600+ triple time of flight (TOF) mass spectrometer was used for LC-MS/MS analysis. The Sciex 5600+ was coupled with a chromXP reversed-phase 3-μm C18-CL trap column  and a nanoViper C18 separation column  in an Eksigent nanoLC (ultra 2D plus) system. A binary mobile solvent system was applied with the following composition: solvent C, 2% (vol/vol) acetonitrile and 0.1% (vol/vol) formic acid in water; solvent B, 98% (vol/vol) acetonitrile and 0.1% (vol/vol) formic acid. To separate the peptides, a 60-min gradient was run with a total run time of 90 min at a flow rate of 200 nL/min, and MS data were acquired in information-dependent acquisition (IDA) with high-sensitivity mode. Each cycle was run for 2.3 s with 250- and 100-ms acquisition times for MS1  and MS/MS  scans, respectively. Every experimental condition was run in quadruplet.(iv) Protein identification and quantification. All raw files (.wiff) were searched in ProteinPilot software  with the Mascot algorithm for protein identification and semiquantitation against the Swiss-Prot 57.15 database . The following parameters were applied to search biotinylated peptides: (i) trypsin as a proteolytic enzyme with a maximum of two missed cleavages allowed; (ii) the allowed tolerance limit for peptide mass error was 20 ppm; (iii) mass error tolerance for fragments was taken up to 0.20 Da; and (iv) carbamido-methylation of cysteine (+ 57.02146 Da), oxidation of methionine (+15.99491 Da), deamination of NQ (+ 0.98416), and biotinylation of lysine (+ 226.07759 Da) were taken as variable modifications. A Pearson correlation plot of peptide intensity was used to monitor data quality between each run and replicate.(v) Data analysis. A protein was considered to be identified if it corresponded to one or more biotinylated peptides, with a posterior error probability (PEP) score of greater than or equal to the median value in the Gaussian smoothing curve of each sample. Note that PEP score refers to the probability that the observed peptide spectrum matches are incorrect. A web-based tool, Bioinformatics and Evolutionary Genomics (http://bioinformatics.psb.ugent.be/webtools/Venn/), was used to identify host proteins that are specific to HEV IRESl RNA and to generate the Venn diagram. The data set with a minimum of 1 unique peptide and a Prot score of 30 or more was considered in the final list of host proteins for further study (er study . Prot schttp://www.unafold.org/mfold/applications/rna-folding-form.php) based on a minimum free energy calculation at 25°C (https://www.gsea-msigdb.org/gsea/downloads.jsp) and Enrichr (https://maayanlab.cloud/Enrichr)   tools.P61247, P62081, P46777, and P61254, respectively. Furthermore, the i-tasser module was used to build their three-dimensional (3D) structures. Based on their c score, the best model was selected and evaluated through Ramachandran plots. Furthermore, the energy minimization of each protein was performed using the AMBER tool. The 3dRNA tool was used to generate the 3D structure of the RNA sequence. For protein-RNA docking, the HDOCK tool was used with the standard cutoffs. In each docking, the top complex was picked for further analysis. The energy-minimized complex structures were used for quantitative and thermodynamic analysis.The protein structures of the four targets RPS3A, RPS7, RPL5, and RPL26 are not reported; therefore, their sequences were retrieved from the “UniProt” by using their ID numbers Renilla-Luc or divided by the percent cell viability, as specified in the Results, and graphs were plotted in GraphPad Prism or Microsoft Excel. Values are shown as mean (±standard error of the mean [SEM]) of three independent experiments performed in triplicate. Western blotting was done as previously described : g1-HEV FP (5′-CGGCCCAGTCTATGTCTCTG-3′), g1-HEV RP (5′-TAGTTCCTGCCTCCCAAAAG-3′), HEV IRESl FP (5′-TATACTCGAGGGTGCCGATCGGTCCC-3′), HEV IRESl RP (5′-TATACCATGGCATCTGGCAGCAAGCTCAG-3′), R-Luc FP (5′-CTTCTTATTTATGGCGACATGTT-3′), R-Luc RP (5′-GCCTGATTTGCCCATACCAATA-3′), control RNA FP (5′-GCCCCCTGGCACATA-3′), and control RNA RP (5′-GAGCGCAGGTTGGAAA-3′).To perform the dual-luciferase reporter assay, HEK293T cells were seeded at 70% confluence, and the next day, 25 nM siRNA was transfected in respective wells using 0.35 μL of Dharmafect transfection reagent, as per the manufacturer’s instructions . Twelve hours after siRNA transfection, the medium was changed, followed by transfection of the dual-luciferase reporter plasmids  using Lipofectamine 2000 at a 1:1 (wt/vol) ratio. Eight hours later, the medium was changed, and cells were incubated for 48 h. Cells were washed with ice-cold PBS and used for luciferase assays using the Dual-Glo luciferase assay kit, following the manufacturer’s protocol . The Firefly-Luc values were divided by those of escribed . Total c2. Next, cells were washed three times with ice-cold PBS (containing 100 μg/mL CHX for CHX treatment), followed by trypsinization and centrifugation for 5 min at 500 × g. The supernatant was discarded, and the cell pellet was resuspended in 1 mL of polysome extraction buffer , incubated on ice for 30 min with occasional mixing, and centrifuged at 12,000 × g for 20 min at 4°C. Total protein and RNA concentration in the clarified lysate were measured by Bradford assay or Nanodrop spectrophotometry, respectively. Aliquots of the lysate were used for cDNA synthesis, Western blotting, and ribosome fractionation.Ribosome fractionation was performed as previously described , 44. HEK2) in diethyl pyrocarbonate (DEPC)-treated water. Then, 2.2 mL of 10% stock solution of sucrose was added to the bottom of a thin-walled, 12.5-mL ultracentrifuge tube, followed by the addition of 2.2 mL each of 20%, 30%, 40%, and 50% sucrose solutions. Tubes were stored at 4°C overnight for linearization. The next day, equal amounts of clarified lysate were loaded to the top of the tubes, and weights were equalized. Tubes were centrifuged in a SW40Ti swinging bucket rotor at 190,000 × g for 90 min at 4°C using a Beckman ultracentrifuge . After centrifugation, tubes were set for fractionation in the automated fraction collection unit, and 24 fractions of 500 μL each were collected. Profiles of RNA in the fractions were captured as an indicator of different ribosome fractions . Alternate fractions (1 to 23) were selected for further analysis of RNA and protein distribution profiles.A linear 10 to 50% sucrose density gradient was prepared for ribosome fractionation 1 day before the polysome fractionation experiment. Stock solutions of 10%, 20%, 30%, 40%, and 50% sucrose were prepared using 2.2 M sucrose and 10× salt solution . Twenty micrograms of total RNA was added to the binding buffer , 20 μL of monoclonal anti-m7G cap antibody , and 1 μL of RNase inhibitor  was added, and samples were incubated overnight at 4°C with continuous mixing. Twenty-five microliters of Protein G Plus agarose beads  were washed twice in binding buffer and added to the samples, followed by a 1-h incubation. Samples were centrifuged at 1,000 rpm for 1 min at 4°C. Supernatant containing unbound RNA was collected, and RNA was extracted using TRI Reagent and stored at −80°C. Beads were washed four times in ice-cold low-salt wash buffer , once in high-salt wash buffer , and twice with Tris-EDTA buffer (pH 8.0). Finally, beads were resuspended in elution buffer (1% SDS and 0.1 M NaHCO3), incubated for 5 min at 4°C on a rocker, and centrifuged at 1,000 rpm for 1 min at 4°C, and the supernatant was collected. RNA was isolated from the supernatant using TRI Reagent, and all samples were simultaneously processed for RT-qPCR.For m7 Huh7 cells were lysed in binding buffer , and clarified lysates were incubated with biotinylated control RNA (control RNA [B+]), nonbiotinylated HEV IRESl RNA (HEV IRESl RNA [B−]), biotinylated HEV IRESl RNA (HEV IRESl RNA [B+]), or mock lysate (cell lysate without RNA) for 1 h on a flip-flop rocker at 4°C. Streptavidin magnetic beads were washed twice in binding buffer and added to the RNA-protein mixture, followed by incubation for 1 h, under similar conditions. Streptavidin-bound RNA-protein complexes were washed two times in ice-cold low-salt wash buffer , once in high-salt wash buffer , and once with Tris-EDTA buffer (pH 8.0). Fifty percent of the beads were resuspended in elution buffer (1% SDS and 0.1 M NaHCO3) and incubated for 5 min at 4°C on a rocker, followed by collection of the supernatant for checking the presence of RNA by formaldehyde-agarose gel electrophoresis. The rest of the beads were mixed with 2× Laemmli buffer and incubated at 95°C for 5 min, followed by collection of the supernatant for Western blotting using various antibodies.For biotinylated RNA pulldown assays, 10P values were calculated by two-tailed Student’s t tests (paired two samples for means).Data are shown as mean ± standard error of mean (SEM) of three independent experiments. PXD031009.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier


RNA-based therapeutics have shown great promise in various medical applications, including cancers, infectious diseases, and metabolic diseases. The recent success of mRNA vaccines for combating the COVID-19 pandemic has highlighted the medical value of RNA drugs. However, one of the major challenges in realizing the full potential of RNA drugs is to deliver RNA into specific organs and tissues in a targeted manner, which is crucial for achieving therapeutic efficacy, reducing side effects, and enhancing overall treatment efficacy. Numerous attempts have been made to pursue targeting, nonetheless, the lack of clear guideline and commonality elucidation has hindered the clinical translation of RNA drugs. In this review, we outline the mechanisms of action for targeted RNA delivery systems and summarize four key factors that influence the targeting delivery of RNA drugs. These factors include the category of vector materials, chemical structures of vectors, administration routes, and physicochemical properties of RNA vectors, and they all notably contribute to specific organ/tissue tropism. Furthermore, we provide an overview of the main RNA-based drugs that are currently in clinical trials, highlighting their design strategies and tissue tropism applications. This review will aid to understand the principles and mechanisms of targeted delivery systems, accelerating the development of future RNA drugs for different diseases. The world has recently witnessed the remarkable impact of messenger RNA (mRNA) vaccines in effectively combating the COVID-19 pandemic, opening a new era of RNA drugs for therapeutic applications. mRNA therapeutics have demonstrated immense potential in the fields of protein replacement therapies, vaccines, and gene editing, as it can regulate gene expression and produce specialized proteins To achieve the intended therapeutic effects, RNA drugs should be precisely delivered to specific organs, tissues, or even cells. Targeted delivery endows several acknowledged advantages. Firstly, delivering RNA cargoes to diseased cells is the prerequisite to realize treatment. Secondly, targeting enables us to address the issue of off-target effects, which often limits RNA therapy by causing toxicity and affecting drug safety as well as efficacy. Precise delivery aims to enhance the bioavailability of RNA drugs at the target site while minimizing their distribution to non-target tissues. Lastly, targeted delivery can lower the required dosage of RNA for administration, improve the biosafety of drugs, and increase the clinical tolerance Despite the enthusiastic potential of RNA-based drugs for targeted treatment of various diseases, multiple obstacles remain to be addressed to fully realize their clinical applications To address the limitations of RNA-based drugs in practical use, a variety of non-viral vectors have been employed for therapeutics delivery. Until now, to develop targeting materials and strategies remains the critical challenge for RNA drug clinical translation. Although several targeted delivery systems have arisen, the guideline behind organ/tissue tropism remains unclear Different RNA drugs demonstrate distinct characteristics and therapeutic mechanisms 7GpppN, can bind to the multi-subunit initiation factor eIF4F to promote mRNA binding with ribosomes and translation of mRNA mRNA is a single-stranded ribonucleotide transcribed from a template strand of DNA, which carries the genetic code and directs the synthesis of proteins. Natural mRNA is composed of the following parts: 5' and 3' untranslated regions (UTRs), 3' poly (A) tail, 5' cap. Among these, UTRs do not encode proteins, but can regulate locations of mRNA and translation efficiency in vitro transcribed (IVT) mRNA is delivered into the desired cells by a suitable vector, such as lipid nanoparticle (LNP), the mRNA is released from the vector. Then the mRNA directs protein synthesis with the help of ribosomes and transfer RNA (tRNA), which can be used for protein replacement to treat protein deficiency (e.g. haemophilia B) or protein malfunction (e.g. muscular dystrophy) Compared to DNA drugs, mRNA does not require entry into the nucleus and can function directly in the cytoplasm. This advantage avoids the risk of gene insertion. Additionally, mRNA has transient expression and will be degraded by enzymes in a limited time after entering the body. This property endows mRNA high safety for therapeutic use + and CD8+ T cells and B cells, killing and eliminating viruses or tumor cells et al. used LNPs to deliver mRNA encoding melanoma-related antigens, which induced a strong immune response and prolonged the survival time of mice bearing melanoma To date, one of the most significant applications of mRNA is to be used as the vaccines in the field of cancer immunotherapy and viral infections. The immune system plays a crucial role in fighting cancer and protecting the body from bacteria invasion. However, tumor-associated cells can secrete a variety of cytokines and chemokines through different mechanisms to inhibit immune system activities Gene editing is another important therapeutic field of mRNA-based drugs, leveraging the development of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)) technology siRNA, also known as short interfering RNA or silencing RNA, is a type of short double-stranded RNA with the length of 21-23 nucleotides containing 2 nucleotides overhang miRNA is a single-stranded non-coding RNA molecule that plays a crucial role in the regulation of gene expression and is involved in a series of essential cellular processes, such as cell proliferation, differentiation and apoptosis in vitro, demonstrating the antisense function of synthetic oligodeoxynucleotides for the first time In 1978, Zamecnik and Stephenson reported the use of synthetic oligodeoxynucleotides to inhibit the replication of virus in vivo. To fully realize the potential of RNA-based drugs for disease treatment, further research and strategies are in urgent demand to overcome the problems associated with targeted delivery.The abundance of RNA species and functions endows huge potential in biological and medical field. However, the efficacy of RNA-based drugs can be affected by the challenge of delivering them to specific organs and sites RNA drugs open an era for treating chronic diseases, nonetheless, their full realization is seriously hindered by lack of targeted delivery technology. Despite the fact that RNA delivery systems have been studied for decades, the precise gene regulation in specific tissues or cells remains a huge challenging. Furthermore, understanding the guideline of organ/tissue tropism of nanoparticles will further the advance of next-generation gene delivery technology, facilitating future RNA-based drug development. Herein, we summarize current progress in targeted RNA delivery, from the point of four key factors: the category of vector materials, chemical structures of vectors, administration routes, and physicochemical properties of RNA vectors Figure .et al. found that ionizable cationic lipids with unsaturated tails could improve the delivery efficiency of LNPs due to the increased lipid fusion ability in vivoLNPs are one of the widely utilized non-viral vectors for delivering RNA-based drugs. Typically, LNPs are composed of four components with distinct functions, including permanently charged cationic or ionizable cationic lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids et al. demonstrated that LNPs encapsulating HNF4A mRNA could attenuate liver fibrosis in preclinical mouse model et al. used a degradable lipid to formulate LNPs to deliver Cas9 mRNA/sgRNA for gene editing in vivo. The results showed that the editing efficiency was highly considerable for transthyretin gene editing in the liver, reducing serum protein levels by over 97% with a single dose Despite some studies show that lipid materials may cause liver or lung injuries and the cytotoxicity of LNPs is related to the dosage of administration and the properties of lipids used, LNPs offer several advantages compared to viral vectors, including ease of production, relatively low immunogenicity, high RNA loading capacity, and flexible design options Despite the great potential in clinical translation, extrahepatic RNA delivery by LNPs remains challenging. Alternatively, polymers provide an option for delivery out of the liver. Polymers are divided into different classifications, such as cationic polymers, zwitterionic polymers, polymeric micelles, and dendrimers To further improve the functionalities of vectors, numerous hybrid copolymers, such as lipid-polymer hybrid nanoparticles (LPNs), graphene oxide (GO)-cationic PEI polymers (GO-PEI complexes), polymer-dendrimer hybrids, were developed et al. utilized chitosan-coated PLGA nanoparticles to encapsulate chemically-modified mRNA encoding human cystic fibrosis transmembrane conductance regulator (CFTR), which was efficiently delivered to lungs of CFTR deficient mice following intravenous and intratracheal administrations et al. modified cationic polymers by zwitterionic phospholipidation to selectively deliver mRNA to the spleen and lymph nodes after intravenous administration in vivo, showing great potential in immunotherapy application et al. designed charge-altering releasable transporters (CARTs) via organocatalytic ring-opening polymerization of various lipids and results demonstrated mixed-lipid CARTs could achieve effective mRNA delivery to B cells and T cells of spleen in vivo, and the delivery efficiency was higher than single-lipid CART Numerous novel polymers have been reported to deliver therapeutic cargoes to special organs, particularly the lungs. For instance, hyperbranched PBAEs were synthesized to enable mRNA delivery to the lung epithelium through inhalation, resulting in consistent and controlled protein production without causing pulmonary or systemic toxicity via ligands, intercellular adhesion molecules on membranes or encapsulated cargo inside the exosomes via B cell-derived exosomes to hepatocytes or macrophages, with higher delivery efficiency and lower cytotoxicity in contrast to regular transfection methods et al. developed a technique to track the exosomes from mesenchymal stem cells of bone marrow (MSC-exo), which could accumulate in mouse brains in different pathological models, including Alzheimer's disease, autism, stroke, and Parkinson's disease. To be more specific, the homing mechanism was driven by inflammation in pathological brains and MSC-exo could be selectively taken up by neuronal cells Exosomes are extracellular vesicles (EVs) with diameters between 30 and 100 nm, and are naturally secreted by cells et al. utilized copper sulfide, which was metabolizable in liver to improve the excretion of Au. And the results showed that the conjugation of copper sulfide and Au could accelerate the excretion of AuNPs in hepatocytes Inorganic nanoparticles (INPs) are synthesized from inorganic particles, biodegradable polycations, and typical inorganic materials such as gold, silica, metallic oxide, and others. Among INPs, gold nanoparticles (AuNPs) have been extensively studied in inorganic chemistry and nanomaterials in vivo, for instance, most LNPs tend to deliver mRNA into the liver. Nonetheless, minimal alteration of chemical structures of vectors in the same class might also achieve the goal of targeting, and the research on exploring targeted material structures is in full swing. Beyond Dlin-MC3-DMA, ALC-0315, and SM-102 approved by FDA for clinical use in LNP formulations, numerous advancements have been made in the development of various lipids, lipidoids, and polymers for delivering RNA to targeted organs. Here, we outline the relationship between the chemical structures of these materials and their specific targeting capabilities for different organs  study. As a result, cKK-E12 LNPs delivered siRNA to liver and silenced Phosphatase and tensin homolog (Pten) in hepatocytes with high selectivity  mice  represent one of the predominant LNP delivery systems for RNA-based drugs. Dong and colleagues designed a series of LLNs with TT3 as the core structure as depicted in Figure r Figure B. Subseqe Figure C. Furthee Figure D 116. Inet al. used linear diacrylate as the backbone and monomer E63 with two secondary amines as the end-cap to synthesize PBAE polymers, achieving preferential expression of mRNA in liver In addition to LNPs mentioned above, several other vectors with different chemical structures can also realize liver-targeting. Rui et al. discovered that the presence of the fifth component, called 'Selective Organ Targeting (SORT)' molecule, could alter the organ-targeting effect of LNPs in vivo. The organ selectivity depended on the type and amount of SORT lipid added , which achieved mRNA expression in hepatocytes, pulmonary endothelial cells, and antigen presenting cells of the spleen in vivo, showing superior delivery efficiency compared to cKK-E12 LNPs  molecules, which targeted spleen for mRNA delivery and translation, demonstrating application potential in immunotherapy and vaccines 10, Dong and colleagues optimized the position of functional groups to obtain the TNT-b10. Further optimizing the formulation of TNT-b10 LLNs resulted in a high signal of mRNA expression in the spleen, which was 10 times higher than that in the liver with intravenous injection et al. combined helper-PBAEs and DOTAP to develop five-element nanoparticles (FNPs) for lung-targeted delivery of mRNA with high stability after lyophilization et al. combined the CD4 antibody with LNPs, achieving much higher accumulation of radiolabeled mRNA in the spleen compared to non-modified LNPs after systemic administration. Further expression of a reporter gene showed CD4-targeted LNPs could specifically deliver Cre mRNA to the spleen and lymph nodes The spleen, which is rich in lymphocytes and macrophages, is an important immune organ. A library of ionizable amino-polyesters were synthesized s Figure B 125. Afs Figure C. Furthes Figure D-E 126. et al.  could co-deliver Cas9 mRNA and sgRNAs with a high efficacy in vitro and in vivo. Specifically, ZA3-Ep10 LNPs enabled gene editing in the liver, kidney, and lungs in vivo in a short time CRISPR/Cas9 gene editing technology has shown revolutionary impact on the gene therapy field. However, delivering the editing tool safely, effectively, and accurately to the target sites remains a significant challenge et al. reported that PBAE DD90-C12-122 could deliver mRNA to the lungs after intravenous administration, and PEGylation increased the delivery efficacy. Further optimizations, such as varying the carbon chain lengths of alkylamine and the molar ratios of diacrylate and amines, would lead to polymeric nanoparticles with greater delivery potential targeting different subtypes of lung. Analysis of the lung cell types after administrating the optimized formulation of polymers showed the transfected cells were mainly among pulmonary endothelial cells and few immune cells et al. synthesized a library of PBAEs and identified two polymers, D90-C12-103 and DD90-C12-103, which used diacrylate-amine as the backbone, could deliver pDNA and mRNA to the lung of mice post systemic administration. Results showed that the fluorescence peak of mRNA was much higher than that of pDNA in vivo after delivery and area analysis of radiance flux and luminescent images at different time points demonstrated that DD90-C12-103 vector was superior to D90-C12-103 for mRNA delivery in vitro and in vivo. A top polymeric vector, PE4K-A17-0.33C12, formulated with 5% F127 and FLuc mRNA was verified to show high luminescence activity in the lung after intravenous administration Researchers have also investigated the relationship between other vector structures and their targeting abilities, beyond LNPs. Kaczmarek To date, the liver, lung, and spleen are commonly targeted organs in current research, however, targeting other organs such as the eyes, skin, heart, and brain holds great potential but remains more challenging in clinical applications.et al. synthesized a series of LNPs with bisphosphonate (BP)-lipid, which showed higher affinity for bone-related fragments in vitro and higher bone-targeting in vivo compared to LNPs without BP-lipid Despite the translation potential, most LNPs tend to accumulate in the liver, and overcoming the natural liver targeting characteristics of LNPs has become a key challenge in nano-vector research. Recently, Sahay group developed LNPs decorated with an oligomer peptide to deliver mRNA to neural retina in rodents and non-human primates after intravitreal administration. This breakthrough demonstrates the potential of LNP-mRNA in the treatment of inherited retinal diseases and represents significant progress in LNP penetrating biological barriers to achieve extrahepatic targeting in vitro. Among them, P1E28 containing alkyl alcohol side chain and piperazine rings with two tertiary amines showed the highest transfected efficiency. In vivo results showed that P1E28 polymeric nanoparticles delivered Cre mRNA to skin dermal fibroblasts with a higher delivery efficiency than endothelial cells, keratinocytes and macrophages. Further mechanism analysis indicated the ability of P1E28 to target skin fibroblasts might be mediated by CD26 and FAP overexpressed in fibroblasts The skin is the largest organ in the human body and skin aging is related closely to the fibroblast in dermis. Recently, Francisco and Ferreira designed hundreds of polymers and selected six polymers with similar chemical structures composed of diacrylate, special amine, and bisacrylamide, which could transfect mouse fibroblasts with high efficiency via intravenous injection. By lipidating the neurotransmitter, the formulated lipid nanoparticles could pass through the BBB by mediating receptors and enter the central nervous system (CNS) to eventually release encapsulated small molecule drugs, macromolecules and gene editing proteins in neuronal cells. It has been demonstrated that the addition of NT1-lipidoids to facilitate vectors penetrating the BBB is suitable for various types of LNPs et al. modified liposomes with cell-penetrating peptides and transferrin ligands, achieving the targeting of brain capillary endothelial cells expressing TfR in vivovia inhibiting the expression of tau protein in a traumatic brain injury mouse model Brain represents another hard-to-target organ, and the presence of BBB limits the therapeutic effect of brain diseases  et al. developed a modular targeting platform called anchored secondary scFv enabling targeting (ASSET), which was a membrane-anchored lipoprotein integrated with LNP and could interact with Fc constant domain of the antibody. The results showed that siRNA-loaded LNPs formed by adding ASSET and RIg could be taken up by targeted cells in vitro and produce desired gene knockdown in vivo. Further research found that altering RIg could achieve targeting of various leukocyte subsets specifically, demonstrating the potential applications of this platform in different disease models et al. utilized the ASSET strategy to connect anti-Ly6c+ mAbs to LNPs, achieving targeted delivery of therapeutic mRNA to Ly6c+ inflammatory leukocytes of mice with inflammatory bowel disease. These results highlight the potential of ASSET in the field of cancers, inflammatory diseases, and rare genetic disorders Cancer is one of the leading causes of death in humans. Targeting drugs provide a precise way to target tumor cells without affecting normal cells, reducing side effects and improving overall survival rates compared to traditional chemotherapeutic drugs Cancer immunotherapy is a highly effective approach to fight against cancer, including using cancer vaccines. However, a major obstacle lies in how to target dendritic cells (DCs) to activate the immune response. In a recent study, RNA-lipoplexes (RNA-LPX) without ligand decoration were designed to target DCs for the delivery of cancer antigens and activation of immunoreaction post intravenous administration. Through adjusting the charge ratio of formulation, the positively charged RNA-LPX primarily targeted the lungs and the fluorescence expression shifted from lung to spleen with a reduction of the cationic lipid content. Notably, in a phase I clinical trial (NCT02410733), three melanoma patients were treated with RNA-LPX encoding the tumor antigens, and the produced IFNα and T-cell responses demonstrated the broad applicability of the RNA-LPX considering that antigens of any tumor cells could be encoded by RNA via intravenous (i.v.) injection or locally administrated via subcutaneous (s.c.) injection, intramuscular (i.m.) injection, intradermal (i.d.) injections, and so on Apart from vector materials, the routes and sites of administration significantly influence the therapeutic outcomes and biodistribution of RNA drugs via the intravenous injection are prone to concentrate selectively in the liver because of its abundance of ApoE, exerting an essential influence on LNP-mediated RNA drugs et al. reported that intraperitoneal administration of LNPs enabled a shift in specific mRNA expression from the liver to the pancreas Intravenous injection is commonly used for LNP-mediated delivery of mRNA or siRNA drugs. This route of administration allows the drugs to reach the target site through circulation. Notably, most LNPs administrated et al. converted LNPs from liver-targeting to lung-targeting et al. developed phosphatidylserine LNPs that could mediate effective mRNA expression in both lymph nodes and spleen after intravenous injection et al. reported a SORT strategy, which achieved the redirection of LNPs to extrahepatic organs by supplementing SORT molecules. Specifically, LNPs were shown to target the spleen by adding anionic lipids and modulating their formulations et al. showed that zwitterionic phospholipidated polymers efficiently delivered mRNA to the spleen, and these polymers with specific amphiphilic and zwitterionic structures exhibited lymph node transfection capability Numerous efforts have been made to systemically deliver RNA to the lung for therapeutic use in pulmonary diseases. For instance, by rationally designing the structure of cationic lipids, Qiu via intravenous administration, inhalation or intratracheal injection via a microneedle-based system and indicated that this technique could be used as a collagen replacement therapy for the treatment of skin diseases and aging via intramuscular injection, enabling the drugs to be concentrated in the draining lymph nodes and trigger a strong immune response from T cells As systemic delivery targets specific organs with intricate difficulties, local administration can be a preferable alternative for certain clinical applications. RNA-based drugs administered by topical delivery are expected to show a therapeutic effect at the specific site. In the case of treating pulmonary diseases, RNA drugs can be administrated to the lung After numerous studies presenting the challenges faced by vectors in delivering RNA, it is evident that the barriers preventing the selective accumulation of drugs may be closely associated with the physicochemical properties of designed vectors via intravenous administration, owing to the adsorption of ApoE by binding to the LDLR that are over-expressed on the hepatocytes Ka values can influence their organ targeting. More specifically, the preferred pKa range for liver, spleen, and lung targeting is 6.2~6.5, 2~6, and 7~9, respectively The formation of protein coronas is believed to be closely associated with the biodistribution of nanoparticles in the body. Upon intravenous injection, the nanoparticles come into contact with the serum, leading proteins to adsorb on their surface and form a distinctive protein corona layer in vivo. For instance, previous studies have revealed that nanoparticles with a diameter of approximately 5 nm are rapidly cleared by the renal filtration process 2), making it an appealing target for RNA therapeutics upon systemic drug delivery. To maximize the drugs' efficacy in the lungs, particles with diameters of less than 3 μm are generally preferred, as they optimize deposition in the alveoli The particle size and the dispersion of delivery vectors play a crucial role in constructing target delivery systems that meet different clinical needs. In order to overcome multiple intracellular and extracellular delivery barriers, attention should be paid to how the vector size affects the bio-interface interactions. Vectors of different sizes present different organ or tissue tropism via the interaction with negatively charged cell membranes. However, high-density surface positive charges can be correlated with corresponding toxicity, thus limiting their application to some extent. In accordance with the desired therapeutic effect, researchers have developed amphiphilic polymers or ionizable lipid nanoparticles that exhibit outstanding endosomal escape efficiency than conventional permanent positive vectors. These new materials are generally neutral under the physiological condition so as to mitigate the toxicity et al. adjusted the lipid charges in LNPs, mediating controlled pKa and specific organ tropism of nano-systems Ka may likewise exhibit a propensity to accumulate in different organ tissues, which is one of the pivotal factors in the design of vectors. Therefore, the rational design of non-viral vectors with desired charges can lead to satisfactory therapeutic effects for RNA-based drugs.Surface and vector charges of nanoparticles are significant physicochemical properties that can be engineered to enhance the site-specific accumulation of RNA vectors and affect their delivery profile Other characteristics, such as particle shape and biodegradability, should also be taken into consideration when designing and evaluating the delivery performance of vectors. Studies have demonstrated that the shape of the nano-vectors plays a crucial role in its delivery journey. For instance, disc-shaped nanoparticles interact more effectively with the vessel wall and exhibit higher specificity for endothelial cells compared to spherical ones Over 20 years have passed since the first RNA-based drug was approved by FDA, and now several RNA drugs have been approved for therapeutic applications in different diseases, including TTR-mediated amyloidosis, duchenne muscular dystrophy, hypercholesterolemia, etc. Additionally, hundreds of candidate drugs are currently under clinical studies. Here, we present a summary RNA-based drugs that have been applied in clinics Table , includiKLKB1 gene, could block production of kallikrein and further reduce bradykinin to treat hereditary angioedema. In clinical I/II trial (NCT05120830), the NTLA-2002 encapsulating Cas9 mRNA and KLKB1-specific sgRNA delivered by LNP resulted durable reductions of kallikrein protein in plasma with well tolerability.mRNA therapy has gained immense popularity since the outbreak of COVID-19. Although no mRNA drug for cancer treatment has been approved by the FDA, there are several mRNA-based therapies for cancer in various stages of clinical trials, which can be divided into two major categories roughly based on the encoded antigens All the mRNA drugs mentioned above utilize LNPs as the delivery vectors, highlighting their significance in the field of RNA-based drugs. However, one exception is AZD-8601 mRNA, which has entered clinical trial (NCT03370887) for heart failure. AZD-8601 mRNA is formulated in citrate buffered saline with biocompatibility and is delivered by epicardial injections to express vascular endothelial growth factor A protein directly. Developed by AstraZeneca and Moderna, this drug increased left ventricular ejection fraction and enhanced heart function compared to placebo group via lactate dehydrogenase (LDH) and excretion of oxalate in urine, which can cause severe kidney injury or kidney failure N-acetylgalactosamine  can achieve liver-targeting and the mechanism is that the GalNAc can combine with asialoglycoprotein receptors (ASGPRs), which are overexpressed by hepatocytes specifically via inhibiting activation of complement component 5. Alnylam declared that in phase II clinical trials (NCT03841448), Cemdisiran reduced the ratio of urinary protein and creatinine (UPCR) by an average of 37% at 24 hour without adverse effects after subcutaneous administration compared to placebo group Most of these drugs approved by FDA target the liver, particularly siRNA drugs. In addition to the five siRNA drugs receiving market authorization, there are also some siRNA drugs in clinical trials and most of them are designed to treat liver-related diseases, such as Nedosiran. Primary hyperoxaluria (PH) is a metabolic disease caused by abnormal liver metabolism, resulting in excessive oxalate production from glyoxylate et al. have demonstrated the powerful effect of CDR132L in large pig animals and the results showed that this drug significantly improved the systolic and diastolic function of the heart and reversed cardiac remodeling after repeated monthly administration via aerosol administration Similar to siRNA, ASO drugs on the market are primarily used to treat patients with rare diseases, but they have a wider range of target organs, including liver, muscle, eyes, and so on. The diseases they address include nervous system diseases, muscle diseases, cardiovascular diseases, and metabolic diseases. In addition to rare diseases, an increasing number of ASO drugs are being developed for other common diseases and are currently in middle- or late-stage clinical trials It is noteworthy that over half of the clinic trials on RNA-based drugs are focused on mRNA, highlighting the huge potential of mRNA therapeutics in RNA-based therapy at present. However, the majority of active clinic trials involving mRNA are derived from COVID-19 vaccines and for the indication of infections. If we exclude these vaccine-related trials, most remaining trials are still in the early-stage Since the discovery of the therapeutic potential of RNA drugs half a century ago, numerous delivery platforms have emerged to enable the safe and efficient delivery of RNA. Through the diligent efforts of researchers in library screening and rational design of delivery systems, several RNA-based drugs have received marketing approval or are currently undergoing clinical trials. As the focus has gradually shifted towards better adapting to clinical needs, efforts are being made to target specific sites for the treatment and reduce the side effects of RNA therapy. With a deeper understanding of the targeting mechanism, researchers are striving to achieve targeted therapies for RNA drugs by using different categories of vectors for delivery, tailoring vector chemical structures, adapting various administration routes, and altering physicochemical properties of vectors. These major advances in delivery systems provide great potential for RNA therapy.Although the first siRNA drug utilizes the LNP vector, the following four siRNA approved drugs instead employ the GalNAc technology, which has become the primary strategy for siRNA-targeted delivery to hepatocytes. While in the field of mRNA-based therapy, advanced LNPs have been developed in two widely inoculated vaccines for COVID-19, known as BNT162b2 and mRNA-1273. This breakthrough has facilitated the exploitation of mRNA-target delivery systems, which have enabled selectivity for organs or tissues by modifying the lipid chemical structures and their formulations. Despite the progress, much work remains to be undertaken to achieve better clinical translation. 1) A deeper understanding of the targeting mechanism is required to develop universal guidelines for achieving targeting to each organ or even different cell subtypes, thus facilitating the design of next-generation delivery systems. 2) Additional investigations on the physicochemical properties, morphology, stability, and other characteristics of the vectors are required to facilitate further targeting and druggability. One can design vectors with specific sizes or charges to achieve targeting delivery of RNA according to the characteristics of different organs and tissues. 3) More attention can be paid to the inherent targeting ability of some natural vectors and figuring out the mechanism of their native tropism, which can guide the future specific targeting. Identifying the exact mechanism of targeting delivery will fundamentally help to design the appropriate vectors to achieve precise delivery. 4) Biocompatibility of vector materials should be considered to reduce toxicity and avoid undesired immunogenicity. 5) Inspired by protein corona-guided and GalNAc-conjugated targeting delivery system, more efforts should be made to screen specific receptors possessed by cell subsets for cellular level targeting. 6) Stepwise targeted drug delivery strategy from organ, tissue, cell to organelle may be utilized to overcome obstacles in delivery process and improve delivery precision.Overall, the targeted RNA-based therapies have already demonstrated tremendous therapeutic potential in numerous clinical applications. As an integral part of RNA therapeutics, the development of a more precise delivery system is of undeniable significance. The prospects are promising that targeted RNA drugs have the potential of utility in diverse disease fields. Although some challenges remain for targeted RNA delivery, the enormous and continuous advances in RNA therapy will undoubtedly improve our health and lives.


Saccharomyces cerevisiae, RNA Pol II transcription termination can follow two different routes: the poly(A)-dependent one used for most of the mRNAs and the Nrd1/Nab3/Sen1 (NNS) pathway for non-coding RNAs (ncRNA). The NNS targets include snoRNAs and cryptic unstable transcripts (CUTs) generated by pervasive transcription. This review recapitulates the state of the art in structural biology and biophysics of the Nrd1, Nab3 and Sen1 components of the NNS complex, with special attention to their domain structures and interactions with peptide and RNA motifs, and their heterodimerization. This structural information is put into the context of the NNS termination mechanism together with possible prospects for evolution in the field.A substantial part of living cells activity involves transcription regulation. The RNA polymerases responsible for this job need to know ‘where/when' to start and stop in the genome, answers that may change throughout life and upon external stimuli. In  Th. Th23]. KDs = 2–15 µM) than Pcf11 (70–160 µM). Rtt103 also binds CTD-pSer2/7 (1.6 µM)  ,40 39,40 6.1 µM) , probabl 6.1 µM) . In Nab3GUAACC and CCGUAGCC RNAs with 2.1 ± 0.1 µM and 8 ± 1 µM KD values [GUAA/CGUAAA/UUUAGUAAUCC confirm the involvement of both RRM and SD in the core GUAA recognition [GUAAA and UUUAGUAAUCC) do not show direct contact with the protein but are loosely stabilized by intra-RNA contacts.NMR and X-ray structures of the Nrd1 RNA-binding domain (RBD) reveal an unusual arrangement  Figure . The canD values , and theognition  , a critical gene linked to neurological disorders and involved in transcription termination and R-loops regulation . Sen1 infication . Sen1 hafication  and can fication . In contfication . The X-rfication . The twofication . In the fication . Biochemfication ,86.In addition to the HD, Sen1 has an N-terminal domain (NTD) that has been involved in interactions with Pol II  and Pol Biophysical and structural data complement genetic and biochemical studies to propose an integrated NNS mechanism . Nrd1/NaNrd1, Nab3 and Sen1 have large IDRs containing short protein binding motifs, large low complexity/prion-like domains and homodimerization motifs . In addiSeveral structural aspects of the NNS complex need to be experimentally addressed in the future; perhaps the most important is related to the structure and interactions of Sen1 N-terminal domain. The versatility of Nrd1 CID in the recognition of non-CTD peptides suggests that Pcf11 and Rtt103 CID might also be able to interact with non-CTD peptides yet to be discovered. Continuing with Nrd1, its particular RBD architecture is conserved in SCAF4 and SCAF8 AlphaFold models, suggesting, that apart from RNA binding, these domains could play a role in other interactions yet to be identified; perhaps with other regions of RNA Pol II?Coming to Nab3, the structure and function of its ABD has not yet been studied. It is tempting to investigate if this domain is involved in the processing of RNA Pol III products.Finally, the importance of Nab3 nuclear granules  deservesThe Nrd1, Nab3, Sen1 (NNS) pathway performs, in budding yeast, transcription termination of ncRNAs like snoRNA and CUTs. Understanding the molecular interactions between its components and with other parts of the transcription machinery is critical to propose mechanistic models.Nrd1, Nab3 and Sen1 are RNA-binding proteins with modular architectures and capability to interact with peptide motifs (i.e. Nrd1-CTD/NIM interactions), heterodimerize (Nrd1/Nab3) or oligomerize (Nrd1/Nab3 PrLDs). Many of these interactions have been structurally and biophysically characterized providing important insight into the NNS mechanism.There are still missing pieces to unravel the NNS structural puzzle, including the structure and interactions of Nab3 ABD and Sen1 NTD domains and the biomolecular condensation properties of Nrd1/Nab3 low-complexity regions involved in the formation of nuclear granules upon stressors.


Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.The mtDNA of the myxomycete  Although most of the genes of eukaryotic organisms are located on nuclear chromosomes, a small number of genes are located on the mitochondrial DNA (mtDNA) in mitochondria. These genes are necessary for mitochondrial function and biogenesis. They include genes that encode mRNAs for protein subunits of complexes of the ETC (electron transport chain) and mitochondrial ATP synthase involved in the OXPHOS (oxidative phosphorylation) pathway. In addition, some of the genes encode tRNAs and rRNAs necessary for mitochondrial protein synthesis and often genes encoding mRNAs for protein subunits for the mitochondrial ribosomes .Rickettsia and the mtDNA of certain protists, exemplified by Reclinomonas americana P. polyceiewed in  in the αum mtDNA ,17,18,19omycetes  and prodial RNAs .P. polycephalum this co-transcriptional mechanism consists of the mitochondrial RNA polymerase proceeding along the template DNA strand, adding templated nucleotides during normal transcription until it arrives at an editing site. The polymerase then pauses in transcription, adds a specific non-templated nucleotide or dinucleotide to the 3′ end of the nascent RNA, and then resumes normal transcription using the DNA template to specify nucleotide addition.The mechanism of this RNA editing is the co-transcriptional addition of specific nucleotides to the 3′ end of the nascent RNA transcript at editing sites by the mitochondrial RNA polymerase ,22. ThisP. polycephalum is similar to other mitochondrial RNA polymerases that lack MICOTREM RNA editing in that it consists of a single polypeptide with significant homology to several bacteriophage RNA polymerases [The mitochondrial RNA polymerase of ymerases . How theymerases  have shoymerases  have proP. polycephalum mitochondrial RNA polymerase is able to add random nucleotides to the 3′ end of RNAs in vitro in the presence of complex RNA sequences. Likewise, Sarcar and Miller [P. polycephalum mitochondrial RNA polymerase can also add specific nucleotides to the 3′ end of RNAs in vitro by limited primer extension has not been determined.A second major unanswered question about MICOTREM editing is how non-templated nucleotides are specified at RNA editing sites. This specificity is clearly necessary since any of the RNA nucleotides can be inserted at RNA editing sites but always the same nucleotide or dinucleotide is inserted at a given RNA editing site. Miller and Miller  have shod Miller  have shoAn alternate way in which the edited nucleotide could be specified is through a binding factor that recognizes the flipped-out nucleotide in an edited RNA/template DNA duplex (see above) and either provides an identical nucleotide triphosphate to the active site of the RNA polymerase which would be added without a template or directly adds the identical nucleotide to the 3′ end of the nascent RNA through a terminal transferase activity. This model would require a specific binding factor for each of the mono- or dinucleotides that can be inserted. Binding of the factor at the editing site might cause the RNA polymerase to pause in transcription to allow the insertion of the non-template nucleotide by either mechanism.Comparison of RNA editing sites within the same genes of different myxomycetes shows that while they all display MICOTREM editing, the location of RNA editing sites varies relative to conserved regions within analogous genes . In contKrishnan et al.  describeEditing site patterns may also be altered by the removal of RNA editing sites. Landweber  and SimpP. polycephalum mtDNA is the presence of 26 open readings that do not correspond to any of the genes classically observed on mitochondrial DNAs [A second unique feature of ial DNAs ,9. Most ial DNAs ,11 The fThe 26 SURFs are interspersed within the classical genes of the mtDNA in four groups. Group 1 SURFs are designed A, B, and C and would be transcribed counterclockwise in The Group 2 untranscribed region has six SURFs, two that would be transcribed clockwise in Flavobacteriales bacterium  recently identified during a metagenomic search of ocean water from the marine abyssalpelagic zone, Pacific Ocean, North Pacific Gyre, Station ALOHA . All three hypothetical proteins have a region of similarity of about 200 amino acids starting at 139 amino acids from the N-terminus. SURF R  has a region of identity to SURF 7  in the mitochondrial mF plasmid (see below). This region of identity in SURF R is 474 nucleotides (158 amino acids) in length starting near the N-terminus. This region of identity is the site of homologous recombination between the circular P. polycephalum mtDNA and the mF plasmid (see below). The one SURF with the potential to produce a protein with a known function is SURF Y. SURF Y has the potential to produce a protein 724 amino acids in length. This protein has significant homology to a number of single subunit RNA polymerases from linear mitochondrial plasmids. This RNA polymerase is presumably not the mitochondrial RNA polymerase used to transcribe genes on the mtDNA of P. polycephalum, since this SURF is not transcribed, and the encoded RNA polymerase is not produced. , located in the mitochondria of some  strains . It cont strains . This li strains . This 77 strains  and may  strains .+  phenotype which allows mtDNA recombination between heteroplasmic zygotes. Its ability to recombine with mtDNA which anciently derives from eubacterial DNA makes it reminiscent of integrating bacteriophages. Bacteriophage phi29 is a member of the invertron family. It has a 19,285 base pair linear double stranded DNA genome, inverted terminal repeats, 5′ terminal binding proteins, and genes for RNA and DNA polymerases similar to linear mitochondrial plasmids, but in addition has genes for structural and capsid proteins [2ββ’σ multisubunit RNA polymerase. The involvement of bacteriophage in the endosymbiont origin of mitochondria is unexplored. Further characterization of the genes encoded in the mF plasmid may provide insights into whether this plasmid has bacteriophage origins. Moreover, the fact that integration of the plasmid occurs in the SURF R region of the mtDNA is further evidence that this region also derives from a mitochondrial plasmid. Further characterization of the SURFs in the mtDNA and linear MF plasmid may strengthen this hypothesis.The origin of the mF plasmid is unclear. It apparently does not have any obligate function since it is absent from some strains without any growth or respiration phenotype. Kawano et al.  report tproteins . Mitocho


ARs, which are stabilized by postsynaptic scaffold proteins and recruited by presynaptic signals. However, how GABAergic neurons trigger signals to transsynaptically recruit GABAARs remains elusive. Here, we show that UNC-43/CaMKII functions at GABAergic neurons to recruit GABAARs and modulate inhibitory synaptic transmission at C. elegans neuromuscular junctions. We demonstrate that UNC-43 promotes presynaptic MADD-4B/Punctin secretion and NRX-1α/Neurexin surface delivery. Together, MADD-4B and NRX-1α recruit postsynaptic NLG-1/Neuroligin and stabilize GABAARs. Further, the excitation of GABAergic neurons potentiates the recruitment of NLG-1-stabilized-GABAARs, which depends on UNC-43, MADD-4B, and NRX-1. These data all support that UNC-43 triggers MADD-4B and NRX-1α, which act as anterograde signals to recruit postsynaptic GABAARs. Thus, our findings elucidate a mechanism for pre- and postsynaptic communication and inhibitory synaptic transmission and plasticity.Disturbed inhibitory synaptic transmission has functional impacts on neurodevelopmental and psychiatric disorders. An essential mechanism for modulating inhibitory synaptic transmission is alteration of the postsynaptic abundance of GABA ARs by triggering anterograde signals.The pre-and postsynaptic communication is critical for faithful synaptic transmission and induction of synaptic plasticity. Here, the authors found that CaMKII functions at GABAergic neurons to recruit GABA ARs). Disturbed inhibitory synaptic transmission has functional impacts on the pathology of neurodevelopmental and psychiatric disorders (including autism spectrum disorders and depression). An essential mechanism for modulating inhibitory synaptic transmission and plasticity is altering the postsynaptic abundance of GABAARs1. Long-term potentiation and inhibition of GABAergic transmission have been associated with increased or decreased GABAAR synaptic abundance, respectively3.Fast synaptic inhibition is mediated by neurotransmitter GABA and GABA-activated chloride channels , including the synaptic adhesion molecule NLG-1/Neuroligin, and the FERM domain-containing protein FRM-318. The postsynaptic GABAARs and inhibitory postsynaptic currents are eliminated in double mutants lacking both NLG-1 and FRM-317.Like other postsynaptic receptors, GABA21. Other reports have shown that GABAergic motor neurons secrete the ADAMTS-like extracellular protein MADD-4B/Punctin, which promotes the localization of GABAARs at inhibitory synapses at NMJs in C. elegans16. In addition, during the induction of activity-dependent synaptic plasticity, the excitation of the presynaptic neurons could trigger the recruitment of postsynaptic receptors and require anterograde signals released by presynaptic neurons. The transsynaptic recruitment of receptors has been extensively studied at excitatory synapses27. However, how the GABAergic neurons trigger anterograde signals to recruit GABAARs remains unclear.Besides the postsynaptic scaffolds, the presynaptic neurons also play essential roles in positioning and clustering postsynaptic receptors. During synaptogenesis, the innervation by presynaptic neurons releases anterograde signals to induce receptor aggregation and clustering in the post-junctional membranes. For example, at mammalian cholinergic synapses, cholinergic motor neurons release extracellular proteoglycan agrin to recruit acetylcholine receptors to postsynaptic membranes2+/Calmodulin and functions as a ubiquitous mediator of cellular Ca2+ signals28. CaMKII is well known as an essential component of postsynaptic density (PSD) proteins at excitatory synapses29, and necessary for NMDA receptor-dependent long-term potentiation (LTP)36. At GABAergic synapses, postsynaptic CaMKII has been reported to phosphorylate GABAARs within the TM3-4 domain to regulate receptor insertion at the cell surface41. CaMKII is required for the induction of rebound potentiation in Purkinje neurons44 and is also required for the moderate N-methyl-Daspartate receptor (NMDAR)-activating stimuli-induced long-term potentiation of inhibition (iLTP) in hippocampal neurons45. Besides that, it has been known for a long time that CaMKII is expressed and functions on the presynaptic side51. However, whether the presynaptic CaMKII could transsynaptically recruit postsynaptic receptors and be involved in inhibitory synaptic transmission and plasticity remain elusive.Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine-specific protein kinase activated by the CaC. elegans NMJs as a model to study how presynaptic neurons trigger anterograde signals to recruit GABAARs at inhibitory synapses. We found that UNC-43, the C. elegans ortholog of CaMKII, is required for GABAARs recruitment and modulates inhibitory synaptic transmission. Experiments using multiple reporter fusion constructs showed that UNC-43 functions at GABAergic motor neurons to recruit GABAARs in the same pathway with NLG-1, but not FRM-3. Next, we demonstrated that UNC-43 promotes presynaptic MADD-4B secretion and cell adhesion molecule NRX-1α/Neurexin GABAergic motor neuron surface delivery. Together, MADD-4B and NRX-1α recruit postsynaptic NLG-1 and stabilize GABAARs. Further, we confirmed that the activity-dependent plasticity in the inhibitory synapses requires the presynaptic UNC-43, MADD-4B, and NRX-1α, and is mediated by the NLG-1-stabilized GABAARs. Collectively, our work elucidates how presynaptic neurons transsynaptically recruit postsynaptic receptors during synaptogenesis and activity-dependent plasticity.Here, we utilize the C. elegans, the majority of GABAergic neurons in the nervous system are inhibitory motor neurons that innervate body-wall muscles55. At the nerve cord, muscle arms elongate and form synapses with GABAergic axon terminals. As a result, the C. elegans body-wall muscles receive direct synaptic inputs from both cholinergic and GABAergic motor neurons56. To visualize endogenous GABAARs, we used a similar strategy as the previous report by inserting a tagRFP coding sequence after the signal peptide sequence of the unc-49 gene that encodes GABAAR in C. elegans using the CRISPR-cas9 genome editing system18. The tagRFP can label all three GABAAR subunits: UNC-49A, UNC-49B, and UNC-49C  as a synaptic model. In 49C Fig. . To testAR abundance, we used the unc-43(js125) null allele58, which deletes 17 kb of sequence positioned downstream of the promoter and the 10th exon and lacks most of the sequence encoding UNC-43  in the unc-43 mutants was decreased by around 35% compared with wild type  and N-AchR (ACR-16) in the unc-43 mutant animals. However, neither of them presented decreased fluorescent intensities compared to the wild type , in cholinergic motor neurons (under the unc-17 promoter), or in body-wall muscles (under the myo-3 promoter) . We found that the decreased synaptic abundance of GABAARs in the unc-43 mutant was partially rescued by either UNC-43D or UNC-43G isoform61 in GABAergic motor neurons; no rescue was observed with the cholinergic neuron or body-wall muscle groups  expression in pan-neurons (under rab-3 or unc-43 promoter), in both GABAergic and cholinergic motor neurons (under unc-25 promoter+unc-17 promoter), and in both GABAergic motor neurons and muscle cells (under unc-25 promoter+myo-3 promoter) in the unc-43 mutants, and we found that the GABAARs puncta fluorescence defects in the unc-43 mutants can be fully rescued by expressing UNC-43 in pan-neurons or in both GABAergic and cholinergic motor neurons, but not with both GABAergic motor neurons and muscle cells groups  using CRISPR-cas9 and analyzed GABAAR abundance. The GABAAR fluorescence intensity was reduced by about 25% in unc-43(k42r)  gain-of-function allele that bears a constitutively activating mutation E108K in the active site core62. The GABAAR fluorescence intensity was significantly increased in the unc-43 mutant  Fig. , indicatant Fig. , supportARs in the unc-43 mutant are correctly localized at GABAergic synapses, we labeled the GABAergic synapses by fusing the endogenous UNC-57/Endophilin in GABAergic motor neurons with GFP in the tagRFP-tagged GABAARs strain using a split GFP complementation system64   Fig. . Furtherons Fig. , indicatARs synaptic recruitment defect in the unc-43 mutants caused impairment of GABAergic synaptic transmission, we patch-clamped body-wall muscles and recorded spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Consistent with the previous report50, we observed a dramatic decrease in mIPSC frequency in the unc-43 mutants  and body-wall muscles (under myo-3 promoter)  Fig. . We founude Fig. . Note thles Fig. . These rARs are compromised in the unc-43 mutants, we recorded the 0.2 mM and 0.5 mM GABA-evoked currents, which were unaltered in unc-43 mutants  in the unc-43 mutant50. This discrepancy likely arises from the different protocols of recording. These results verified that UNC-43 is required for both GABAAR synaptic recruitment and inhibitory synaptic transmission.To investigate whether the expression and/or surface delivery of GABAARs in the unc-43 mutant is the secondary result of synaptic structural defects, we measured the puncta fluorescence and intensity of endogenous UNC-57/Endophilin in GABAergic motor neurons in the unc-43 mutant. We observed no significant decrease in puncta fluorescence intensities and densities between wild-type and unc-43 mutants , snb-1 mutants, unc-13 mutants (which lack synaptic vesicle release), unc-31 mutants (which lack dense-core vesicle release), and egl-3 (which lack neuropeptide maturation)  gain-of-function mutation was eliminated in the nlg-1 mutant, but not in the frm-3 mutant  in the muscle cells of wild-type and unc-43 mutant worms. The GABAARs recruitment defects of nlg-1 mutants were rescued by the transgene expression of the C-terminal GFP-tagged NLG-1 in the muscle cells  gain-of-function mutation is not able to increase the GABAAR puncta fluorescence intensity in the madd-4b; nrx-1 double mutants , and the MADD-4B-GFP fusion protein is able to rescue the GABAARs recruitment defects in the madd-4b mutants  kinase-dead mutants  mutants  in the GABAergic motor neurons (under unc-25 promoter) and delivering pulsed blue-light excitation , and it lasted within 2 h   Fig. , supportAR puncta fluorescence at synapses afterward in the unc-43 mutant animals, and restoring UNC-43 expression in GABAergic motor neurons rescued the activity-dependent increase of GABAAR recruitment -activating stimuli cause CaMKII selectively translocating to inhibitory synapses to phosphorylate GABAAR β3S383 and recruit scaffold protein gephyrin, which promotes GABAARs accumulation and immobilization at synapses70. Besides, an acute increase in neuronal activity of cultured hippocampal neurons also promotes the phosphorylation of β3S383 by CaMKII39. Further, in cerebellar somatodendritic basket cell synapses, CaMKII is required for the rebound potentiation (RP) by phosphorylation of GABAARs receptor44.Most previous studies of CaMKII have focused on its facilitation of AMPAR recruitment to enhance synaptic strength at postsynapses51. CaMKII is enriched at presynaptic sites, and accounts for ~2% of the total synaptic vesicle protein amount71. Presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 was shown to block excitatory synaptic plasticity in cultured hippocampal neurons47 and presynaptic CaMKII was implicated in synaptogenesis and synaptic transmission at NMJs of both Drosophila and C. elegans50. In the present study, we identified a transsynaptic GABAAR recruitment function of presynaptic CaMKII, thus demonstrating CaMKII’s involvement in inhibitory synaptic transmission. We also show that this transsynaptic recruitment activity is supported by MADD-4B secretion and NRX-1α surface delivery and propose the hypothesis that UNC-43-triggered anterograde signals are required for the activity-dependent plasticity at inhibitory synapses. Thus, the function of CaMKII in recruiting postsynaptic GABAARs is conserved in species, and both presynaptic and postsynaptic CaMKII can recruit postsynaptic GABAARs.However, many studies have indicated that CaMKII has functional impacts at presynapsesAR abundance defects, MADD-4B secretion defect, and NRX-1α motor neuron surface delivery were similar in the unc-43 null mutant as in worms expressing a kinase-dead mutant unc-43 (k42r)  Fig.  and 7. TARs are stabilized by distinct synaptic scaffolds at C. elegans NMJs: there are NLG-1-stabilized GABAARs and FRM-3-stabilized GABAARs18. It has been shown that the NLG-1-stabilized GABAARs and FRM-3-stabilized GABAARs together mediate inhibitory synaptic transmission18. However, it remains unclear which type of receptors mediate inhibitory synaptic plasticity. Here, by using the nlg-1 and frm-3 mutants, we demonstrate that NLG-1-stabilized GABAARs mediate activity-dependent plasticity at inhibitory synapses. Further, our study indicates a plausible explanation for how NLG-1-stabilized GABAARs are recruited to inhibitory postsynaptic elements upon excitation of presynaptic GABAergic motor neurons: it is likely that Ca2+ influx after GABAergic motor neuron excitation activates UNC-43, which can trigger more MADD-4B secretion and NRX-1α surface delivery. These molecules can subsequently act as anterograde signals to recruit additional NLG-1 to postsynaptic elements to somehow help stabilize the GABAARs. Given that no physical interactions have yet been reported between NLG-1 and GABAARs, the nature of this stabilization remains unclear. A recent study proposed that FRM-3 and CASK ortholog LIN-2 mediate the interaction between NLG-1 and GABAARs18. However, an additive defect in GABAARs recruitment and inhibitory synaptic transmission was observed in the frm-3; nlg-1 double mutant17. Further, UNC-43 functions in a single pathway with NLG-1 to recruit GABAARs, but not with FRM-3. In summary, the current data does not support the conclusion that NLG-1 recruits GABAARs through FRM-3.Previous studies have demonstrated that GABAFurther information and requests for resources and reagents should be directed to and fulfilled by the Lead Contact Xia-Jing Tong (tongxj@shanghaitech.edu.cn).C. elegans strains were derived from the wild-type Bristol N2 (Caenorhabditis Genetics Center) strain. Worms were cultivated at 20 °C on nematode growth medium (NGM) plates seeded with Escherichia coli (E. coli) under standard conditions. The OP50 strain of E. coli was used as a food source for all experiments, except where the HB101 strain was utilized for the electrophysiology study. The well-fed young-adult hermaphrodites were used in all experiments except for coelomocyte imaging experiment in which the adult day-5 animals were used. Transgenic animals were prepared by microinjection, and integrated transgenes were isolated following UV irradiation or by single-copy insertion mediated by miniMos or CRISPR-cas9. A complete list of the strains used in this study can be found in Supplementary data All unc-43 mutants. The js125 mutation is a deletion from 10 kb upstream of the transcription initiation site to exon 1058. The kinase-dead mutant unc-43(k42r) was generated by CRISPR-cas9, which caused the K42R coding variant. The gain-of-function unc-43 (n498) mutation is a point mutation that causes single amino acid substitution (E108K) and makes the kinase partially active even in the absence of Ca2+.Molecular information on madd-4B mutants. The xj0739 mutation is a C insertion in the first exon of madd-4B, introducing an early stop codon and generating a 21 amino acids (aa) product.Molecular information on The constructs used and created in this study are detailed in Supplementary data The primers used in this study are detailed in Supplementary data The reagents, software, and algorithms used in this study are detailed in Supplementary data tagRFP-T-unc-49(xj1024) knock-in was generated based on the CRISPR-cas9 technique previously described72. The tagRFP-T with 3x GS linker was inserted between Gln23 and Asp24, and an extra Gln was added before Asp24 to guarantee the UNC-49 signal peptide coding sequence integrity. The sgRNAs were designed on CRISPR (http://crispor.tefor.net/)73. The single-stranded repair template was generated by overlap extension PCR (purified and heated at 95 °C for 5 min). To remove off-target mutations, the transgenic worms were outcrossed with N2 and used for further studies.The unc-43(k42r) allele was also generated by CRISPR-cas9. A 99 bp single-stranded repair template was synthesized and purified before injection.Similarly, the unc-43(xj764[LoxP::unc-43p::unc-43::LoxP]) allele was generated for unc-43 conditional knockout. The first LoxP with EcoRI restriction enzyme cutting site was inserted upstream of unc-43 promoter (3 kb before exon 1 of UNC-43 transcripts K11E8.1a to K11E8.1 l), and the second LoxP with EcoRI was inserted into the first intron. Under the action of Cre recombinase driven by the tissue-specific promoter, about 3800 bp genomic DNA containing the promoter and 59 bp coding sequence of UNC-43 transcripts (K11E8.1a to K11E8.1l) including start codon was tissue-specifically removed. For the transcript K11E8.1r, the deletion of the 59 bp coding sequence causes a frameshift and an early stop codon. myo-3 and unc-25 promoters were cloned into pFX_NLS::Cre vector74. The conditional knockout animals were verified by PCR and sequencing.The unc-57::GFP11*7(xj1544) and GFP11*7-unc-43(xj1455) allele was generated for tissue-specific labeling UNC-57 and UNC-43. unc-57::GFP11*7(xj1544), seven copies of the GFP11 coding sequence were inserted at the C-terminus of the unc-57 genomic locus. Briefly, GGGS (linker)-GFP11*7 coding sequence was inserted before the stop codon of unc-57 isoforms (T04D1.3a to T04D1.3d). For GFP11*7-unc-43(xj1455), seven copies of the GFP11 coding sequence were inserted at the N-terminus of the unc-43 genomic locus. Briefly, GGGS (linker)-GFP11*7-GGGS (linker) coding sequence was inserted after the start codon of unc-43 isoforms (K11E8.1b to K11E8.1l). In parallel, the GFP 1-10 fragment was constitutively expressed in the GABAergic motor neurons. As a result, the endogenous UNC-57 and UNC-43 localized in the GABAergic neurons can be visualized.The N2 worms were injected with 10 ng/μL of PCFJ910 plasmid-of-interest containing the promoter and open reading frame (contains a Neomycin resistance gene), 50 ng/μL of pCFJ601 (Mos1 transposase), 10 ng/μL of pGH8 (Prab-3::mCherry), 2.5 ng/μL of pCFJ90 (Pmyo-2::mCherry), and 5 ng/μL of pCFJ104 (Pmyo-3::mCherry). Animals were grown under 25 °C after injection. Neomycin (G418) was added to plates 24 h after injection at a final concentration of 1.5 μg/μL. Homozygous animals with the desired insertion were verified by PCR and sequencing.xjSi0009 allele encodes GFP-tagged MADD-4B driven by GABAergic motor neuron-specific unc-25 promoter. The GFP sequence was inserted after Phe711 of the MADD-4B coding sequence.The xjSi0016 allele encodes dual-tagged NRX-1α driven by the unc-25 promoter. The pHluorin sequence was inserted between Ile29 and Ile30 of NRX-1α, and the wrmScarlet was inserted after Val1540.The 75. The mean fluorescence intensities of reference FluoSphere microspheres  were measured during each experiment that controlled for changes in illumination intensities. Automatic image analysis was performed as in previous reports76. Briefly, four image parameters were defined: (1) Peak (Fpeak): Absolute peak fluorescence, is the averaged ratio of peak fluorescence to the bead standard of the cord analyzed; (2) Cord (Fcord): absolute axon fluorescence, which is the ratio of baseline fluorescence to the bead standard; (3) Puncta F: peak-to-cord magnitude, 100X (Fpeak – Fcord)/Fcord; (4) Density: is the number of peaks found per 10 μm of cord analyzed. All fluorescence values are normalized to wild-type controls to facilitate comparison. To assess the synaptic accumulation of fluorescent proteins, we used the Puncta F: peak-to-cord magnitude.For UNC-49, UNC-29, UNC-57, UNC-43, FRM-3, and NLG-1 puncta fluorescence imaging, images were captured using a 100x objective (NA = 1.4) on an Olympus microscope (BX53). For ACR-16, MADD-4B, and NRX-1α puncta fluorescence imaging, images were captured using a Nikon 60×1.4 NA objective on a Nikon spinning-disk confocal system (Yokogawa CSU-W1). Young-adult worms were immobilized with 30 μg/μl 2,3-Butanedione monoxime (Sigma). The maximum intensity of dorsal cord projections of Z-series stacks was obtained by Metamorph software (Molecular Devices). Line scans were analyzed in Igor Pro (WaveMetrics) using a custom scriptARs, the mean of Pearson’s coefficients between the distribution of fluorescence intensity in each channel were compared in Figs. For colocalization assay, images were captured using a 100x objective (NA = 1.4) on an Olympus microscope (BX53). The maximum intensity of dorsal cord projections of Z-series stacks was obtained by ImageJ. The background was removed using the Subtract Background plugin of Image J . The fluorescence intensity along the cord was evaluated with the Plot Profile plugin (line width 20 pixels). For each channel, the values were normalized to the value of maximal intensity. To assess the correlations between GABAergic boutons and GABAFor coelomocytes imaging, images were captured using a Nikon 60 × 1.4 NA objective on a Nikon spinning-disk confocal system (Yokogawa CSU-W1). 5-day adult worms were immobilized with 30 μg/μl 2,3-Butanedione monoxime. All fluorescence values are normalized to wild-type controls to facilitate comparison. To assess fluorescence intensities of coelomocytes, region of interests (ROIs) were traced for the coelomocytes and a background area outside of the animal in ImageJ. Maximum intensities of these ROIs were exported, and fluorescence intensity was corrected for background intensity.ARs was assessed and analyzed by Chi-square test.Super-resolution images were captured with a Nikon 60 × 1.4 NA Objective on a Nikon spinning-disk confocal system (Yokogawa CSU-W1 SoRa) based on SoRa mode. Live young adult animals were anesthetized with 30 μg/μl 2,3-Butanedione monoxime (Sigma) and the regions of dorsal cords were excited by a 561 nm laser . The maximum intensity of dorsal cord projections of Z-series stacks was obtained by ImageJ. The number of animals with diffusing or punctate GABA77. Briefly, 1 mM aldicarb was added to the NGM plate. More than 20 animals at the young-adult stage were picked on each plate. The paralyzed animals were counted every 10 min. At least three double-blind replicates were performed for each genotype.The aldicarb assay was performed as previously describedC. elegans as previously described17. Worms were superfused in the extracellular solution  at 22 °C. Whole-cell recordings were carried out with the internal solution . The solution was adjusted to pH 7.2 using CsOH) at 0 mV for mIPSCs.Electrophysiology was conducted on dissected For GABA-activated current recordings, 0.2 or 0.5 mM GABA was pressure ejected for 1.0 s onto body muscles of adult worms.E. coli culture and spotted on 3.5 mm NGM plates. Plates were allowed to dry for 24 h before use. L4 transgenic worms with channelrhodopsin variant CHIEF expressed in the GABAergic motor neurons were transferred to ATR plates and grown overnight. Young adult worms were received pulsed blue-light excitation  at 20 Hz for 30 min, and subjected to GABAARs puncta fluorescence analysis or electrophysiology.To prepare the standard agar plates for optogenetic activation of GABAergic motor neurons, 1.6 mM of all-trans retinal  or ethanol (control) was mixed with OP50 t-test  with a scatter plot. Data were analyzed with Prism 8.0 . The Kolmogorov–Smirnov test was performed to determine whether the data were normally distributed. For comparison of the two groups, a two-tailed unpaired Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary filesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary