Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research. Apple hammerhead viroid  -G82/G81 RNA transcripts were modified to 5′-p, 5′-OH, and 5′-cap [mppp(5′)] .Modification from 5′-ppp to 5′-p in the 5′-terminus of the RNA transcripts was conducted using RNA 5′ polyphosphatase , as described in previous studies . This in2, 0.1 mM ZnCl2, pH 6.0 (New England Biolabs), 40 U RNase inhibitor, and 50 U Antarctic phosphatase, followed by inactivation of Antarctic phosphatase via incubation for 5 min at 70 °C. Both of the modified RNA transcripts (5′-p and 5′-OH) were recovered by phenol-chloroform extraction and ethanol precipitation.Modification from 5′-ppp to 5′-OH in the 5′-terminus of the RNA transcripts was conducted by dephosphorylation with Antarctic phosphatase . This included the incubation of 10 µg of the RNA samples for 1 h at 37 °C in a 100 µL reaction mixture containing 50 mM Bis-Tris-Propane HCl, 1 mM MgCl2; 2 mM spermidine; 10 mM dithiothreitol; 2 mM each of ATP, UTP, and CTP; 0.2 mM GTP; 0.5 mM cap analog m7G(5′)ppp(5′)G (Invitrogen); 40 U of RNase inhibitor; and 50 U T7 RNA polymerase (Invitrogen). Subsequently, 2 μL of 20 mM GTP was added to the reaction mixture to a final concentration of 2 mM. After incubation for 50 min at 37 °C, the plasmid DNA template was digested with 5 U of RNase-free DNase I (Takara Bio) at 37 °C for 15 min.Co-transcriptional capping of the 5′-terminus was performed as described in previous studies . First, Modification from 5′-ppp to 5′-cap  in the 5′-terminus of the RNA transcripts was conducted using the Vaccinia Capping System (New England Biolabs). For this, 10 µg of RNA samples were denatured at 65 °C for 5 min in 15 µL of ultrapure water, placed on ice for 5 min, and then incubated for 1 h at 37 °C in a 20 µL reaction mixture containing 1× capping buffer (New England Biolabs), 0.5 mM GTP, 0.1 mM SAM, and 1 µL Vaccinia Capping Enzyme. The co- and post-transcriptional capping reaction mixture volumes were then increased to 100 μL with ultrapure water; thereafter, 10 μL of a solution containing 5 M ammonium acetate and 0.1 M EDTA (pH 8.0) was added to the reaction mixture. The reaction products were purified by phenol-chloroform extraction, followed by precipitation with an equal volume of 2-propanol.Recovered RNAs were resuspended in 11 µL of the RNA storage solution (Ambion) and quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Each RNA sample (0.5 µg) was checked by electrophoresis in a 1.2% denaturing agarose gel containing 2.2 M formaldehyde using 1× MOPS running buffer, as described above.Solanum lycopersicum) cultivar “Rutgers” was inoculated with PSTVd and TCDVd; the tomato cultivar “Newskij” was inoculated with CSVd; and the cucumber (Cucumis sativus) cultivar “Suyo” was inoculated with HSVd. The inoculated plants were grown under greenhouse conditions of 24–28 °C  or in a growth chamber at 24 °C (CSVd). The infectivity of inoculum nucleic acids was evaluated by the extent of infection in inoculated plants. First, 5-fold serially diluted nucleic acids were used as an inoculum, and the inoculated plants were investigated by RT-PCR at 42- or 56-days post inoculation (dpi). Afterwards, the extent of infection in plants inoculated with a certain amount of RNA transcripts was investigated every 7 days to identify the approximate days required for the detection of a viroid by RT-PCR.Plasmid DNAs or RNA transcripts were suspended in inoculation buffer . Thereafter, the inoculation suspension (1.25−1.5 µL) was rubbed onto carborundum (600 mesh)-dusted cotyledons (two cotyledons per plant) using a finger covered with a disposable latex finger cot. At least three seedlings at the cotyledon stage were used for each inoculation with DNA or RNA. The tomato . The sequences of PSTVd, TCDVd, HSVd, and CSVd were amplified using their respective primer pairs PSTV-7P and PSTV-7M [A previously described method was used to extract total nucleic acids from approximately 50–100 mg upper uninoculated leaf tissue using potassium ethyl xanthogenate  with som PSTV-7M  (for PST PSTV-7M  (Table S PSTV-7M  (Table Sw/v) agarose gel containing formaldehyde at a final concentration of 0.66 M in 1× MOPS buffer. The fractionated RNA was transferred from the agarose gel to a nylon membrane  by capillary transfer and hybridized with a DIG-labeled cRNA probe specific for PSTVd at 55 °C, as described in previous studies [The accumulation of PSTVd in tomatoes inoculated with the different transcripts of PSTVd was compared by Northern blot hybridization. Total RNA was individually extracted using RNAiso Plus (Takara Bio) from the upper uninoculated leaves of five tomato plants 14 dpi. Subsequently, equal quantities of total RNA from the five plants were pooled and subjected to Northern blot hybridization, as described in previous studies . The poo studies . The hybWe inoculated plants with 2, 0.4, and 0.08 µg of plasmid DNA containing monomeric viroid cDNA along with the duplicated 11-nt sequence and monomeric or dimeric linear viroid RNA transcripts. Inoculated plants were assayed for infection at 42 dpi for PSTVd and TCDVd and 56 dpi for HSVd and CSVd. The infectivity of PSTVd-Int-G96/G95 and TCDVd-Ca-G97/G96 monomeric RNAs was higher than that of pUC9 inserted with monomeric cDNA of PSTVd and TCDVd, respectively. In addition, the infectivity of PSTVd-Int-G96/G95 and HSVd-citV1(270G)-G82/G81 monomeric RNAs was equal to or higher than that of PSTVd and HSVd dimeric RNAs, respectively . FurtherThe relationship between the putative processing/ligation site and the infectivity of viroid RNA transcripts was further investigated by comparing the time-dependent infection rate. In this experiment, monomeric (1 µg each) and dimeric (2 µg each) RNAs of PSTVd and HSVd were inoculated into “Rutgers” tomatoes and “Suyo” cucumbers, respectively. The reason is that one molecule of dimeric viroid RNA produces one molecule of monomeric viroid RNA by in vivo processing; therefore, 2 µg of dimeric viroid RNAs were inoculated, based on the number of monomeric RNA molecules that would be generated. Starting from 14 dpi, the extent of infection in the inoculated plants was investigated every seven days using RT-PCR. At 14 dpi, with respect to the infection rate of PSTVd, it was observed that PSTVd-Int-G96/G95 RNA had the maximum infectivity, followed by PSTV-Int-G16/U15, PSTVd-Int-2U and PSTVd-Int-G265/C264 RNAs. Moreover, the first infection by the PSTVd-Int-G265/C264 inoculum was confirmed seven days after the confirmation of infection with the other three inoculums. However, by 42 dpi, the infection with PSTVd-Int-G265/C264 RNA was eventually confirmed in all five tomato plants; this pattern showed a 14- or 21-day delay in comparison to the pattern of infectivity of PSTV-Int-G16/U15 and PSTVd-Int-2U RNAs or PSTVd-Int-G96/G95 RNA . IncidenTo understand the infectivity of precisely monomeric viroid RNA transcripts possessing 5′-ppp, the 5′-ppp termini of PSTVd and HSVd monomeric RNA transcripts were modified to 5′-p, 5′-OH, or 5′-cap. Subsequently, each of these modified RNA transcripts was used as an inoculum at 0.4 µg per plant, except 2U PSTVd RNA, which was inoculated at 0.8 µg per plant. The 5′-capped transcripts were prepared using the following two procedures. Co-transcriptional capping is usually considered to produce a mixture of capped and uncapped  transcripts, whereas post-transcriptional capping produces nearly 100% capped transcripts. Starting from 14 dpi, the extent of infection in the inoculated plants was investigated every seven days using RT-PCR. Unexpectedly, at 21 dpi, a cucumber plant inoculated with 5′-OH monomeric HSVd RNA was the first to exhibit HSVd infection. Thereafter, at 28 dpi, three cucumber plants inoculated with 5′-ppp monomeric HSVd RNA and one cucumber plant inoculated with 5′-p monomeric HSVd RNA became positive for HSVd infection; however, cucumbers inoculated with co- and post-transcriptionally synthesized 5ʹ-capped HSVd RNA were still negative for HSVd infection. Eventually, by 35 dpi, all inoculated cucumbers were confirmed to be infected by HSVd .In the first inoculation experiment, all the tomato plants inoculated with the five different monomeric transcripts as well as one dimeric transcript of PSTVd were positive for PSTVd infection at 14 dpi, as detected by RT-PCR; these results were probably obtained due to the optimal plant growth conditions, such as abundant natural sunlight . Since tWe developed an in vitro transcription system for synthesizing a complete monomeric linear viroid RNA without any non-viroid sequence and with the ability to infect host cells from a cDNA clone. The infectious monomeric viroid RNA transcripts can serve as a powerful and useful tool for viroid studies. The infection efficiency of monomeric RNA transcripts initiated at the putative processing/ligation site was apparently higher than that of the pUC9 inserted with the corresponding monomeric cDNAs, and it was either equal to or more than that of the dimeric RNA transcripts . HoweverIn addition to the transcripts mentioned above, monomeric RNA transcripts initiated at other guanine nucleotides distinct from the putative processing/ligation site were also found to be infectious ; these rIn addition to the infectivity of monomeric PSTVd and HSVd RNA transcripts bearing the 5′-p terminus, infectivity was identified in the monomeric PSTVd and HSVd RNAs bearing the 5′-ppp, 5′-OH, and 5′-capped termini; these results are consistent with those from previous reports ,24,26. TAutographa californica nuclear polyhedrosis virus BVP (baculovirus protein phosphatase) [E. coli RppH  [Caenorhabditis elegans PIR-1 [Although a linear RNA bearing 5′-ppp and 3′-OH termini cannot be circularized directly, all the seven monomeric RNA transcripts of four viroids were infectious to host plants . Additiophatase) , E. colidH/YgdP) , human PdH/YgdP) ,45, and ns PIR-1 . HoweverPrevious studies have reported that the infectivity of co-transcriptionally capped monomeric CSVd RNA is the same as that of uncapped CSVd RNA, although the transcribed RNA includes a non-viroid sequence . IncidenSimilar to the co-transcriptionally capped RNA products, the 5′-OH RNA products may include a small amount of 5′-ppp RNA; therefore, the infectivity of the 5′-OH RNA product may be due to the presence of the 5′-ppp RNA. Eventually, all plants inoculated with the 5′-OH RNA were infected with PSTVd or HSVd; therefore, it cannot be denied that there is a possibility that the 5′-OH termini of monomeric RNA transcripts may be phosphorylated with a low efficiency by an unknown mechanism in vivo. Another possibility is that the 3′-OH terminus may be phosphorylated to 3′-P, and subsequently, it may be converted to 2′,3′>P terminus. It has been previously reported that an RNA ligase purified from wheat germ is capable of ligating PSTVd linear molecules that have 5′-OH and 2′,3′>P termini, but it is incapable of ligating PSTVd linear molecules that have 5′-P and 3′-OH termini . Based uIn conclusion, the precisely monomeric viroid RNA transcripts bearing 5′-ppp and 3′-OH are infectious to their host plants. Our in vitro transcription system of producing an infectious monomeric linear viroid RNA from a cDNA clone is convenient, and it will facilitate further viroid research since in vitro mutagenesis can be rapidly conducted by a simple procedure of inverse PCR, self-ligation, and cloning. Moreover, we recorded infectivity in monomeric RNA transcripts that were initiated at the guanine nucleotide identical to as well as distinct from the putative processing/ligation site, thereby suggesting the applicability of this system for studying viroids whose processing/ligation site is other than a guanine nucleotide.


TOMM40) gene may contribute to the risk of Alzheimer’s disease (AD). Currently, there is no consensus as to whether TOMM40 expression is up- or down-regulated in AD brains, hindering a clear interpretation of TOMM40’s role in this disease. The aim of this study was to determine if TOMM40 RNA levels differ between AD and control brains. We applied RT-qPCR to study TOMM40 transcription in human postmortem brain (PMB) and assessed associations of these RNA levels with genetic variants in APOE and TOMM40. We also compared TOMM40 RNA levels with mitochondrial functions in human cell lines. Initially, we found that the human genome carries multiple TOMM40 pseudogenes capable of producing highly homologous RNAs that can obscure precise TOMM40 RNA measurements. To circumvent this obstacle, we developed a novel RNA expression assay targeting the primary transcript of TOMM40. Using this assay, we showed that TOMM40 RNA was upregulated in AD PMB. Additionally, elevated TOMM40 RNA levels were associated with decreases in mitochondrial DNA copy number and mitochondrial membrane potential in oxidative stress-challenged cells. Overall, differential transcription of TOMM40 RNA in the brain is associated with AD and could be an indicator of mitochondrial dysfunction.Increasing evidence suggests that the Translocase of Outer Mitochondria Membrane 40 ( APOE) which has an odds ratio of 3.7. The strength of the association between APOE and AD risk is orders of magnitude larger than all other AD loci combined, suggesting that this locus is a major biological contributor to the risk of AD. Therefore, deciphering the mechanistic role of the APOE locus in AD should provide insight into the etiology of this devastating disease.Understanding the role that genetics plays in the pathogenesis of AD has been a major research focus for the past three decades. These collective efforts have provided valuable insights into the molecular mechanisms associated with this disease. The advancement of genome-wide approaches has led to the identification of more than 40 AD-associated genetic loci. However, most of these loci have only moderate effect sizes with odds ratios ranging from 1.1 to 1.5 (AlzGene), except for the apolipoprotein E gene  to strongly associate with AD  -1. The calculated amplification efficiency is as follows: 0.92 ; 0.90 (TOMM40 IVS9); 0.89 (pseudogene P1b/P2); 0.91 . For each sample, qPCR assays were performed in triplicate. Information on primers, probes, and TaqMan assays is listed in TOMM40 RNA quantification, all reactions were quantified by using a fixed threshold (0.15) in the linear range of amplification and recording the number of cycles  required for the fluorescence signal to cross the threshold. To control for the quantity of input RNA, we quantified ACTB mRNA as an internal control for each sample and obtained a normalized ΔCT value: mean of CT triplicate (target)–mean of the ACTB CT triplicate. In this setting, smaller ΔCT values indicate higher RNA transcription levels. Additionally, fold change (FC) of TOMM40 transcription levels of AD to Control subjects was computed as FC (AD) = 2−ΔΔCt, where ΔΔCT = mean ΔCT (AD)– mean ΔCT (Control) [RT-qPCR assays were performed as previously reported . BrieflyControl) .P1b/P2 primer set was used to measure levels of pseudogene RNAs and IVS9 primers were used to amplify TOMM40 surrogate RNA by qPCR. A primer set spanning Ex4 and Ex5 was used for measuring levels of the total TOMM40 RNA pool. These primers were also used for RT-qPCR (SYBR) assays as listed in TOMM40 RNA pool. For the quality control of QIAcuity dPCR, we replicated the assay with different amounts of template input and showed the reproducibility of the fraction of the target RNA in the total TOMM40 RNA pool.The 2O2, (Sigma) in growth media. For controls, the same number of cells were plated and cultured without H2O2. Cells were collected 24 h post-treatment, subjected to genomic DNA and total RNA isolation, followed by measurement of MtDNA copy number and RNA transcription levels. Three to four independent treatments with H2O2 were performed.Twenty-four hours prior to treatment, the cells were seeded at a density of 70–80%. For RNA transcription and mitochondrial DNA (MtDNA) copy number assays, cells were seeded on a 6-well plate, whereas a 96-well plate was used for the mitochondrial membrane potential assay. We searched the literature for the effects of hydrogen peroxide on mitochondrial function. Based on previously published conditions, we tested multiple concentrations  of hydrogen peroxide in the cell lines and selected 500 µM as an optimal concentration that maintained good cell viability and had noticeable effects on mitochondrial function. The seeded cells were then treated with 500 μM Hydrogen peroxide, HHGB (Hemoglobin), were run separately with 10 ng of DNA in a 384-well optical plate. Each reaction was run in triplicate on QuantStudio 5 . The 10 μL reaction included 10 ng of DNA, 5 μL of 2x Power SYBR Green PCR Master Mix , and 0.05 μM of each forward and reverse primer. Thermal cycling profile consisted of 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles of 15 s at 95 °C, 60 s at 56 °C, and 60 s at 72 °C. The ΔCT method was used to control for the quantity of input DNA for each sample by quantification of HGB DNA. The normalized ΔCT value was calculated: mean of MtDNA CT triplicate– mean of the HGB CT triplicate. The fold change (FC) of the MtDNA copy number isolated from H2O2-treated cells to untreated cells was computed as FC (treated) = 2-ΔΔCT, where ΔΔCT = ΔCT (treated) − ΔCT (untreated) [Reactions for MtDNA copy number count and single copy reference gene, treated) .2O2 for 24 h and then assayed for MMP measurements. A quantity of 2 μM JC-1 was added and incubated at 37 °C, 5% CO2 for 30 min. The reaction plate was washed with PBS and the fluorescence was measured with 488 nm excitation and green (529 nm) or red (590 nm) emission using SpectraMax M2 plate reader (Molecular Devices). All procedures were performed according to the manufacturers’ protocols. Fold change (FC) of membrane potential of H2O2-treated cells to untreated cells was computed as FC (treated) = ratio of red to green (treated)/ratio of red to green (untreated). Three independent MMP assays were performed for each H2O2 treatment for each cell type.MMP of the human cell lines was analyzed using a MitoProbe JC-1 assay kit (Thermo Fisher). The cationic dye, JC-1 , exhibits potential-dependent accumulation in mitochondria, which is indicated by a fluorescence emission shift from monomeric green (529 nm) to JC-1 aggregates red (590 nm). Consequently, MMP change in response to cellular stimuli is represented by the ratio of red to green fluorescence intensity. The membrane potential disrupter, CCCP (carbonyl cyanide 3-cholorophenylhydrazone), was included in all assays as a control to confirm that the JC-1 response is sensitive to changes in membrane potential. Twenty-four hours prior to the hydrogen peroxide treatment, cells were seeded at a density of 70–80% on a black 96-well plate with a clear bottom. The seeded cells were treated with 500 μM HT values and was calculated as follows, ΔCT value = mean of CT triplicate (target)–mean of the ACTB CT triplicate. Statistical analyses were performed using independent samples t-test using the Statistical Package for the Social Sciences (SPSS) version 19 (SPSS). The MtDNA copy number assay and membrane potential assay were not performed in the same cell setting because the membrane potential assay needs to be conducted on live cells. For this reason, data were not statistically compared.The qPCR data is expressed as normalized ΔCTOMM40 mRNA in human brain and its association with AD risk have not been fully established. Studies have reported conflicting results of either up- or down-regulated TOMM40 mRNA in AD brains. The measurement of a gene’s mRNA levels should be a straightforward procedure unless other complications confound this measurement; apparently, an unknown barrier exists in the measurement of TOMM40 mRNA. To expose this obstacle, we first inspected the specificity of TOMM40 cDNA by aligning this sequence to the human genome (hg38) using the Blat tool in the UCSC genome browser . Besides TOMM40 itself, the alignment showed six additional hits, including one known gene (TOMM40L) with moderate homology to TOMM40 and five loci with a high degree of sequence homology. All five of these highly homologous loci contain a TOMM40 cDNA-like sequence lacking either TOMM40 introns or a full open reading frame, classic characteristics of a pseudogene. When queried the public databases, we found established pseudogene records for four of the five loci in the HUGO gene database  with the designated nomenclatures of TOMM40P1, P2, P3, and P4. We designated the unnamed pseudogene “TOMM40P1b” due to its proximity to TOMM40P1. The genomic location, span, and similarity to TOMM40 cDNA of these pseudogenes are listed in The transcription levels of TOMM40 pseudogenes can be transcribed into RNA. To address this question, we developed new RT-PCR assays designed to specifically amplify each pseudogene’s putative RNA transcripts and generated pseudogene-specific DNA templates to serve as positive controls. To generate pseudogene templates, we PCR amplified each pseudogene’s genomic region using primer sets . Due to high homology between P1b and P2, as seen in the phylogenetic tree . We then designed allele-specific PCR primers that carry the unique nucleotide(s) of each pseudogene at the 3′-end of the primers . Because the expected amplicons of pseudogene RNAs can also be generated from pseudogenes’ corresponding genomic DNA, we performed DNase digestion for all RNA isolations and included RT-negative controls for all experiments. In these experiments, we also integrated a TOMM40 cDNA control that was RT-PCR generated using TOMM40 cDNA-specific primers. We then performed RT-PCR reactions using total RNA isolated from these cells. The capillary gel electrophoresis showed that all pseudogenes-specific amplicons were amplified in all cell lines tested, with no amplification from RT-negative controls  and exons 3-4 (Ex3-Ex4)  C. When semplates . This reTOMM40 gene expression assays likely include both RNA species of TOMM40 and its pseudogenes. We then designed experiments to further examine this possibility. Because exact primer and probe sequences of the commercial assays are not publicly available, we generated a TOMM40 cDNA assay that spans splice junctions of exons 4-5 (Ex4-Ex5), the general area targeted by a popular TOMM40 TaqMan expression assay . When tested using end-point PCR, this assay cross-amplified all pseudogene templates  and quantified the cDNA targets using digital PCR (dPCR). From each subject, two amplicons (P1b/P2 and Ex4–Ex5) were quantified separately, and the absolute quantification count of P1b/P2 was then divided by the count of Ex4–Ex5 to generate the fraction. The result showed that P1b/P2 RNA constituted around 10–18% of the total TOMM40-related RNA pool  transcription level is expected to be only a fraction of the spliced mRNA transcription level, this proportion is likely retained consistently across samples obtained from same cell/tissue-types. Thus, an RT-PCR assay based on this principle could provide an accurate surrogate measurement of the actual mRNA transcription level, which can then be used to compare samples from human subjects.The results described above prompted us to conclude that in order to eliminate cross-amplification of TOMM40 using the Ensembl RNA track in the UCSC genome browser. This track shows the presence of six variants of TOMM40 RNA transcripts (T difference) between AD and control frontal lobes . No differences in TOMM40 mRNA transcription levels were observed between AD and control when the commercial assay was used. On the contrary, TOMM40 mRNA showed significantly  higher expression (≈2.5-fold) in AD compared to control when the IVS9 assay was used  scattered across TOMM40  and in rs741780  and compared TOMM40 RNA levels, MtDNA copy number, and mitochondrial membrane potential. After H2O2 treatment, TOMM40 surrogate RNA levels were increased  and a decreased membrane potential  with TOMM40 cDNA. We were even more surprised to find that all five pseudogenes produce RNA transcripts in various human cell lines and PMB tissues. Currently, the biological functions and/or consequences of these TOMM40 pseudogene RNAs are undefined. The TOMM40 pseudogenes are scattered across the human genome with unique flanking DNA sequences; thus, each of them is likely independently regulated. It is plausible that some of these RNAs can serve as templates to produce small peptides, but, to our knowledge, this has never been investigated. Another potential function of these TOMM40 pseudogene RNAs is to regulate the transcription of TOMM40. Carrying highly similar sequences, these RNAs could compete with TOMM40 mRNA for binding to proteins in transcription or post-transcription machineries. Potentially, these pseudogene RNAs could provide an RNA buffer in response to various stress/stimuli, and this buffer effect might minimize severe fluctuations in TOMM40 RNA production. Indirect evidence supporting this concept comes from our experiment of comparing TOMM40 cDNA assays. The commercial TOMM40 assay, which measures both TOMM40 mRNA and pseudogene RNAs, has much tighter transcription levels across the PMB samples when compared to the IVS9 assay that specifically measures TOMM40 RNA only. One explanation is that this commercial assay congests all the TOMM40-related RNAs together, which provide a cushion effect to reduce the variability of single RNA measurement. The estimated fraction of pseudogene RNAs in the total TOMM40-related RNA pool is approximately 10–18% using a single pseudogene assay (P1b/P2) that cross-amplified four out of five pseudogenes. If the transcription levels of all five pseudogenes can be precisely measured, the overall fraction is likely to be significantly higher than this estimation. These results raise an interesting question: why does TOMM40 gene expression need to be rigorously regulated or guarded by such a complex system from a whole genome setting?To determine the source of inconsistent TOMM40 mRNA. This challenge prompted us to develop an alternative approach, which targets the primary RNA transcript of TOMM40 and eliminates undesired co-measurement of pseudogene RNAs. The major difference between the primary transcript and mRNA lies in the RNA splicing. The splicing efficiency depends on the splicing kinetics, transcriptional and splicing regulators, transcription rate, intron length, exon position, RNA structure, and chromatin signatures, including histone marks and DNA methylation [TOMM40 transcription levels, which were measured by the primary transcript-targeted assays (Ex6-IVS6 and IVS9), had a similar profile with ≈2 ΔCT value separating AD and control PMB samples. This result indicated that both measurements were consistent across samples and were suitable to serve as surrogate measurements of TOMM40 mRNA. Between the two assays, the IVS9 has higher transcription levels  when compared to Ex6-IVS6. This difference could be due to either the slower splicing kinetics of IVS9, or simply the presence of a TOMM40 mRNA transcript (ENST592434) that retains the entire intron 9 in its mRNA structure.Due to the cross-amplification of pseudogene RNAs, the conventional RT-PCR assays cannot provide an accurate measurement of hylation ,66. It hhylation ,68. Due TOMM40 RNA is roughly 2.5-fold higher in AD compared to the control frontal lobe. Our results are opposite to prior published studies showing that TOMM40 RNA is downregulated in AD blood [TOMM40 regulatory pathways between blood and the CNS, it also suggested that the transcription levels of TOMM40 pseudogene RNAs could vary across different tissues. Increased TOMM40 RNA transcription in AD brains could be a consequence of prolonged mitochondrial dysfunction, which triggers a feedback response to upregulate structural proteins  to compensate for the compromised mitochondrial function. The upregulated TOMM40 RNA in AD brains has the same trend as the upregulation of APOE mRNA in AD brains compared to control [TOMM40 and APOE in AD brains makes the concept of co-regulation of these genes through the same topological associating domain even more appealing. Whether the upregulation of TOMM40 RNA is truly associated with AD risk remains to be further validated with a larger sample size and across different brain regions. Nevertheless, this study provides a new molecular tool for measuring TOMM40 RNA, making future expanded studies feasible.After resolving the complication of pseudogene RNAs co-measurement, we demonstrated that the surrogate transcription level of AD blood ,36,37,38TOMM40 RNA transcription levels with either APOE rs429358 variants (C/ε4+ vs. C/ε4−) or TOMM40 rs2075650 variants (G+ vs. G−), two SNPs that have been consistently linked to AD risk through GWAS studies. This lack of association suggested that these two genetic variants may not have a direct impact on the increased TOMM40 RNA levels in AD. On the contrary, the SNPs rs10527523 and rs741780 showed significant allelic differences associated with TOMM40 RNA transcription. Because these two SNPs are located between introns 6 and 8 of the TOMM40 gene, this region of TOMM40 might contain functional regulatory elements that influence the transcription of TOMM40. In the case of rs10524523, we observed a higher transcription level  of TOMM40 RNA in the “S” variant when compared to the “VL” allele. This result is opposite to the study of Linnertz et al., who showed TOMM40 mRNA levels were lower in “S” homozygotes compared with “VL” homozygotes in the AD brain [TOMM40 pseudogene RNAs transcription levels could account for these conflicting results.Our genetic association analyses revealed no difference between TOMM40 RNA transcription levels in human cell lines. MtDNA copy number is a measure of the number of mitochondrial genomes per cell and is a proxy for mitochondrial function [TOMM40 RNA levels are associated with a lower MtDNA copy number and a lower mitochondrial membrane potential, which together signified a decrease in mitochondrial function. This observation could be explained by a feedback response to restore mitochondria function via upregulation of TOMM40 mRNA. As a translocase of outer mitochondrial membrane [TOMM40 RNA transcription associated with AD could lead to changes in mitochondrial protein import, which might affect maintenance of mitochondrial membrane potential and overall mitochondrial function.Mitochondria are involved in several cellular functions and are essential for energy production; they are the main organelles that provide energy for brain cells. Indeed, neurons are particularly sensitive to changes in mitochondrial function , and mitfunction ,72,73. Sfunction . Studiesfunction ,76. Mitofunction ,78,79. Tfunction . Our anamembrane , TOM40 pTOMM40 RNA transcription levels with high specificity and sensitivity, circumventing the unintentional co-measurement of TOMM40 pseudogene RNAs. This assay enabled us to accurately investigate the RNA transcription profile of TOMM40 associated with AD. The PMB work showed that TOMM40 mRNA is upregulated in AD vs. control frontal lobe. The ex vivo cultured cell line work showed that upregulation of TOMM40 RNA is likely associated with compromised mitochondrial function. Although future work using a larger sample size is needed to replicate these results, this work pioneers a valuable blueprint to assess APOE-independent effects of TOMM40 in AD risk. Our findings define a new paradigm of TOMM40 gene regulation and provide novel insight into the transcriptional pathway of TOMM40. This pathway involves not only the production of multiple TOMM40 mRNA species, but also a pseudogene-imparted transcriptional program. Many epidemiology studies strongly support the idea that TOMM40 contributes to healthy aging. Because age is the most important known risk factor for AD, it raises an interesting question: could the incidence of AD be a byproduct of a compromised longevity pathway that is carefully guarded via TOMM40-imparted mitochondrial function?Here, we developed a novel assay to measure true


We also touch on how these RNA modifications affect treatment of HNSCCs.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer malignancy worldwide and is known to have poor prognosis. The pathogenesis behind the development of HNSCC is not fully understood. Modifications on RNA are involved in many pathophysiological processes, such as tumor development and inflammation. Adenosine-related RNA modifications have shown to be linked to cancer and may play a role in cancer occurrence and development. To date, there are at least 170 different chemical RNA modifications that modify coding and non-coding RNAs (ncRNAs). These modifications affect RNA stability and transcription efficiency. In this review, we focus on the current understanding of the four major RNA adenosine modifications (N Head and neck squamous cell carcinoma (HNSCCs) mainly occurs in the mucosal epithelium of the oral cavity, pharynx or larynx. It is the sixth most common cancer worldwide, with over 800,000 cases diagnosed annually and an increasing incidence . Exposur6A methylation, m1A methylation, A-to-I RNA editing (New messenger RNA (mRNA) transcripts require additional processing and modifications before translation and protein synthesis. RNA modifications regulate most steps of gene expression, from indirectly controlling DNA transcription through transcription factors, to directly affecting mRNA translation . Many RN editing  and APA  editing . We also6A is the most abundant and well-defined internal modification in mRNA. Methylation of the sixth nitrogen atom of RNA base A affects RNA stability and translation efficiency , methyltransferase-like 14 (METTL14), WTAP, RNA-binding motif protein 15 (RBM15), RNA-binding motif protein 15B (RBM15B), ZC3H13 and KIAA1429.mficiency . This mo6A modification process undergoes first-order catalysis involving the two methyltransferase complexes (writers) METTL14 and METTL3. METTL3 coverts adenosine to m6A through its methyltransferase domain, and METTL14 is responsible for the recognition of RNA substrates. ZC3H13, RBM15 and VIRMA are also incorporated into the methyltransferase complex to regulate METTL14 and METTL3 functions.The m6-methyladenosine on RNA is reversible through coordination of methyltransferases and demethylases. Fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) have been identified as members of non-heme Fe (II)/α -ketoglutarate-dependent dioxygenases. AlkB homolog 3 (ALKBH3), another member of the family, preferentially acts on m6A in tRNAs. The mechanisms where m6A methylation selectively and dynamically targets specific regions of the transcriptome are not fully understood. It is anticipated that additional m6A demethylases will be discovered.The deposition of N6A modifications are unique recognition elements that bind proteins to readers and drive biochemical processes that occur in labeled RNA. The RNA-binding domain or the YTH domain contains YTH-domain proteins 1 and 2 family members . More recently, other readers have been identified, including insulin-like growth factor 2 mRNA-binding proteins  and heterogeneous ribonucleic proteins (HNRNPC and HNRNPA2/B1). These proteins are highly expressed in different cancers and are involved in various molecular mechanisms. These proteins are not always dependent on the recognition of m6A.m1A is a reversible modification produced when a methyl group attaches to the N1 position of adenosine. TRMT61A, TRMT61B, TRMT10C and TRMT6 have the ability to generate m1A modifications. It carries a positive charge when in physiological conditions and blocks the Watson–Crick interface, alters the structure of RNA, regulates protein-RNA interactions and affects the tertiary structure of ribosomes and translation (1A in tRNAs and rRNAs is significantly greater than the abundance of mRNAs. Most m1A sites are located in the 5′-untranslated region (5′-UTR) of RNA (mnslation . In mamm) of RNA . This mo1A “writers” contain a methyltransferase complex, including TRMT6, TRMT61A, TRMT10C and TRMT61B  or coding regions of different lengths are generated through alternative polyadenylation (APA) when poly (A) tails are added or removed to different sites on RNA  Figures. DiffereAPA can affect the transcription in multiple ways . First, The proximal poly (A) site is used to form mRNAs with short 3 UTRs in highly proliferating cells . StudiesEpigenetic and post transcriptional mechanisms play important roles in gene expression and normal physiology editing is a post-transcriptional mechanism that changes the sequence of transcriptional RNA through post transcriptional modifications .in vitro and is specifically expressed only in brain tissue, which may explain why it is thought to be catalytically inactive  where adenosine (A) forms inosine (I) through hydrolytic deamination . A-to-I inactive .6A methylation modifications play key roles in the occurrence and development of carcinomas. Most studies indicate that an increase of m6A methylation levels promotes tumor growth, development and treatment resistance , which promotes the overexpression of KIAA1429 mRNA. Therefore, the methylation level of KIAA1429 (VIRMA) may be closely related to HNSCC  promotes the degradation of BZLF1 and BRLF1 by recruiting the RNA degrading proteins ZAP, DDX17 and DCP2. YTHDF1 ultimately inhibits EBV infection and lytic replication. This process is dependent on XRN1. Xia et al. speculated that EBV introduces m6A modifications into host cells to destabilize BZLF1 and BRLF1, thereby inhibiting lytic replication and maintaining the incubation period of the virus . These modifications on TFRC mRNA have also influence tumor occurrence and proliferation. At the same time, YTHDF1 and iron-related genes (FTH1 and TFRC) are significantly upregulated in tumor tissues , erasing m6A methylation in new FOXM1 and NANOG transcripts and causes chemotherapy resistance , erasers (ALKBH5 and FTO), and readers  were found to be significantly increased in HNSCC tissues , lung cancer, colorectal cancer and pancreatic cancer , erasers  and readers , were significantly changed in all gastrointestinal cancers analyzed.Zhao et al. comprehensively analyzed TCGA data for diagnosed with esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD), pancreatic adenocarcinoma (PAAD) and colorectal adenocarcinoma (COAD)). Expression of m1A regulatory genes are significantly higher in HCC than in normal tissues. In addition, the expression levels of TRMT6, TRMT61A and TRMT10C, as well as ALKBH3 and YTHDF2, are higher in patients with late-stage tumors (G1-G3)  . Shi et rognosis . Meanwhiectively . This isdicators . Based oer cells . Therefo1A regulatory genes in PAAD are associated with cancer stage. Some of these genes serve as writers or readers and have a high mutational frequency. CNV has also been found to have a high frequency of mutations. Meanwhile, changes in the YTHDF1 and TRMT61A genes were found in two PAAD samples. Even though high expression levels of YTHDF1 and YTHDF2 were found to be associated with the poor prognosis of PAAD patients  and several single proteins. CPSF1 is the largest subunit of the protein complex  that recPABPN1 may also act as the core factor of APA regulation. Poly (A) tails grow to ∼250 nucleotides. PABPN1 binds to poly (A) and breaks the connection between CPSF and poly (A) polymerase, thereby controlling the length of the poly (A) tail . Xiang eIn addition, we believe that some oncogenes alter the length of 3′-UTR through APA events, thereby evading gene suppression and promoting the growth of HNSCC . It has Currently, there are only a few studies directly investigating A-to-I RNA editing in HNSCC. One reason for this may be due to limited detection technology in identifying A-to-I editing sites. Previous studies have shown A-to-I RNA editing (R334G) in the tumor suppressor gene prox1 present in 4 of 8 esophageal cancer cases, indicating that A-to-I RNA editing may be closely related to the progression of this cancer . HochberADAR1 may be closely related to the occurrence and development of HNSCC. Zhang et al. showed that ADAR1 expression is related to STAT1, STAT2 and IRF9, and the abundance of ADAR1 protein is related to the activation of JAK/STAT pathway induced by type I interferons. The activation of JAK/STAT pathway regulates the expression of ADAR1, which leads to an abnormal RNA editing spectrum in ESCC . InteresBehm et al. found that mice do not survive to adulthood in the absence of the active editing enzymes ADAR1 or ADAR2. As neurons mature, the number of interactions between ADAR2 and nuclear isomerase Pin1 increases, which contributes to the stability of ADAR2 protein and helps mouse neurons develop and mature . TerajimThe biological role of ADAR3 has been studied over recent years. Wang et al. found that ADAR3 binds to two activity-dependent immediate-early genes in response to brain stimulation that encode the 3′-UTRs of the mRNAs early growth response 1 (EGR1) and bispecific phosphatase 1 (DUSP1), thereby regulating the transcription levels of DUSP1 and EGR1. This result suggests that ADAR3 may play a new role in brain function . ADAR3 wp = 0.04)  cases that combines platinum, fluorouracil and cetuximab chemotherapy . This ca = 0.04) , but theHuR (ELAVL1) is an RNA-binding protein that plays a positive role in regulating tumor survival and invasion. Wang et al. proposed that knockout of HuR through CRISPR/Cas9 (HuR-CRISPR) inhibits tumor progression. Multifunctional nanoparticles can achieve targeted delivery of HuR CRISPR and epirubicin, and significantly improved the symptoms of mice bearing SAS tumors . HoweverLiu et al. proposed that the expression of PD-L1 in HNSCC cytology samples is highly consistent with matched histological samples (Programmed cell death protein-1 (PD-1/CD279) is an important immune checkpoint expressed by T cells. It can be coupled with the programmed death ligand PD-L1 (B7-H1/CD274) expressed by tumor cells and inhibit the activation of T cells and the anti-tumor response, evading immune system clearance. Immune checkpoint inhibitors (ICIs) can block the inhibitory immune checkpoint pathway, thereby reactivating anti-tumor immune activity. Common ICIs include PD-1/PD-L1 inhibitors that can specifically bind to PD-L1 on tumor cells and block the inhibitory immune checkpoint pathway . TIGIT i samples . Lee et  samples . Therefo+ and CD8+ T cells in the blood and tumors  The number of RNA editing changes. An increase in the number of RNA edits can be observed in some cancers, but it does not cause overexpression of oncogenes. The may be due to the presence of these editing sites in normal tissues as well . 2) So fsistance . This mosistance . 3) Evensistance . For exasistance . IGFBP7 sistance ; 4) A-tosistance . For exasistance .In summary, recent studies have revealed RNA editing events in HNSCC. However, there is still very little known about the specific mechanisms of RNA modification to control cancer progression and drug resistance. Currently, investigators have been able to study the editing of non-coding regions using high throughput sequencing technology. At the same time, they have also found that non-coding regions have the highest levels of complexity. As a result, we started to explore the expression regulation of miRNAs and lncRNAs from a new perspective. This new concept breaks traditional thinking, which believes that unedited sequences or biomarkers must be linked to downstream targets through certain mechanisms. The mechanism by which RNA editing regulates the occurrence and development of cancer is still unclear. Research on the pathophysiological functions of RNA modifications in cancer is still in the early stages of research and there is still a long way to go. However, there is increasing evidence that the dysregulation of RNA editing central mediators (such as ADAR) contributes to the progression of cancer. Therefore, future work should focus on how to translate these modifications into available treatment options and how to relate these modifying behaviors to diagnosis and prognosis.


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Due to the lack of potent diagnosis and prognosis biomarkers and effective therapeutic targets, the overall prognosis of survival is poor in HCC patients. Circular RNAs (circRNAs) are a class of novel endogenous non-coding RNAs with covalently closed loop structures and implicated in diverse physiological processes and pathological diseases. Recent studies have demonstrated the involvement of circRNAs in HCC diagnosis, prognosis, development, and drug resistance, suggesting that circRNAs may be a class of novel targets for improving HCC diagnosis, prognosis, and treatments. In fact, some artificial circRNAs have been engineered and showed their therapeutic potential in treating HCV infection and gastric cancer. In this review, we introduce the potential of circRNAs as biomarkers for HCC diagnosis and prognosis, as therapeutic targets for HCC treatments and discuss the challenges in circRNA research and chances of circRNA application. Hepatocellular carcinoma (HCC) is ranked as the sixth most common neoplasm and the third leading cause of cancer-related death worldwide . CurativCircular RNAs (circRNAs) are a novel class of non-coding RNAs generated from back-splicing of pre-mature transcripts by forming covalently closed loop structures without 5′-caps or 3′-polyadenylated tails , 8 Fig, accumulRecent emerging evidence has demonstrated that circRNAs are closely associated with tumor initiation and progression –20. FirsEarly diagnosis is critical for the improvement of HCC prognosis and outcomes. The serum alpha-fetoprotein (AFP) , alpha-fZhang et al. demonstrated that circRNA_104075 was positively regulated by HNF4a, a HCC-promoting transcription factor, and highly expressed in HCC tissues and serum samples. CircRNA_104075 showed greater predictive performance  for HCCs than AFP , AFP-L3  and DCP  .By comparing the serum circRNA profiles between 36 healthy controls, 60 chronic hepatitis C (CHC) and 68 HCC patients, Matboli et al. found up-regulation of hsa_circ_000224 and down-regulation of hsa_circ_001565 and hsa_circ_000520 in the serum samples of HCC patients compared to those in healthy controls and CHC patients . The preBy analyzing differentially expressed circRNAs (DECs) in plasma between 40 healthy controls and 71 HCC patients, Sun et al. recently identified a three-circRNA signature, including hsa_circ_0004001, hsa_circ_0004123, and hsa_circ_0075792, which are highly expressed in HCC patients . This 3-Comparison of circulating circRNAs, in 5 HCC patients before and after hepatectomy, 5 HBV-positive chronic hepatitis (CH) patients and 5 healthy controls, identified that the serum levels of hsa_circ_0009582, hsa_circ_0037120, and hsa_circ_0140117 were high in HCCs and decreased after hepatectomy . Moreovein vitro and in vivo (Down-regulation of hsa_circ_0001445 (circSMARCA5) in HCCs was identified in 3 cohorts of 208 pairs of HCC and corresponding adjacent noncancerous liver (ANL) tissues, and circSMARCA5 overexpression inhibited HCC growth and metastasis in vivo . By compin vivo . It wortin vivo .vs. 0.790 (p=0.036) in the training set , AUC 0.843 vs. 0.747 (p=0.011) in Validation Set 1 , and AUC 0.864 vs. 0.769 (p<0.001) in Validation Set 2    containing three circRNAs , which had greater accuracy than AFP in distinguishing individuals with HCC from Non-HCC . The perRecurrence occurs in more than 70% HCC patients post curative surgery . Even foHsa_circ_0000267 was up-regulated in HCC tumor tissues and positively associated aggressive clinicopathological features such as tumor size and TNM stage . MultivaChromosomal amplification at 7q21–7q31 was closely related to tumor recurrence in various types of cancers including HCCs –62. By aSun et al. have demonstrated that hsa_circ_0027345 (circLRIG3), a nucleus-enriched circRNA, was highly expressed in HCCs and positively associated with aggressive clinicopathological features, such as tumor size, vascular invasion, Edmondson’s grade and later TNM stage, and was identified as an independent risk prognostic factor for OS in HCC patients .The down-regulation of hsa_circ_0001727 (circZKSCAN1) in HCCs were determined by two independent groups , 66. ExpIn addition to serving as a non-invasive biomarker, decreased expression of circSMARCA5 in HCC tumor tissues was significantly correlated with poorer tumor differentiation, more advanced tumor stage, larger tumor size and presence of microvascular invasion. Multivariate analysis demonstrated that circSMARCA5 expression level was an independent risk factor for OS and RFS in HCCs .A recent study has demonstrated that an up-regulated plasma circRNA, hsa_cic_0005397, was positively correlated with tumor size (p = 0.020) and TNM stage (p = 0.006) . MeanwhiHowever, due to the limited sample size and the lack of multicenter validation, the diagnosis and prognosis potential of those circRNAs should be further determined in the future.Like many other non-coding RNAs, such as lncRNAs and miRNAs, circRNAs have been demonstrated to function as either oncogenes or tumor suppressors in HCCs.By comparing circRNA expression profiles in primary HCCs with or without post-surgery pulmonary metastasis, Hu et al. identified that hsa_circ_0085616 (circASAP1) is highly expressed in HCCs with greater metastatic potential . MechaniThrough bioinformatics analysis and experimental validation, Li et al. demonstrated that up-regulation of hsa_circ_0074854 (circMAT2B) in HCCs was correlated with poor prognosis and could serve as an independent prognostic factor . By funcNR2F6 to initiate NR2F6 expression  was highly expressed in HCCs and promoted proliferation, migration and invasion in HCC cells by acting as a protein recruiter to recruit TIP60 onto the promoter region of pression .in vitro and in vivo  was significantly decreased in HCCs and correlated with poor prognosis of HCC patients . CircMTOin vivo .in vitro and in vivo (FMBP), an RNA binding protein, circZKSCAN1 prevented the binding of FMBP with cell cycle and apoptosis regulator 1 (CCAR1) mRNA and in turn inhibited CCAR1 expression and CCAR1-mediated activation of the Wnt/β-catenin signaling pathway, resulting in compromised stemness in HCC cells , an up-regulated circRNA in HCCs, could serve as a sponge for miR-605 to induce FOXO3-mediated ABCB1 expression, resulting in HCC oxaliplatin resistance . CircRNAFor sorafenib-mediated targeted therapy, Wu et al. have identified thousands of deregulated circRNAs in sorafenib-resistant HCC cells compared to sorafenib-sensitive cells . Yang etFor PD1 antibody mediated immunotherapy, circMET has been demonstrated to confer HCC cells resistance to anti-PD1 treatment by enhancing the immunosuppressive tumor microenvironment . Mechanivia regulating the miR449a-MET pathway  of recorded circRNAs   were curP. falciparum and S. cerevisiae  were identified from the great circRNA popularity, whether circRNAs are a group of functional non-coding RNAs is still an open question. The existence of numerous circRNAs in species deficient with RNAi and miRNA pathways raises the question whether as miRNA sponges is the general role of circRNAs or just is an exception. Although some studies have demonstrated that the formation of circRNAs depends on the KA, X-DW, YF, XL, YM, JL, XW, and QH performed extensive literature search and discussion. KA and X-DW drafted the manuscript. XK, JG, and HW edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China , the Scientific Program of Shanghai Municipal Health Commission (SHWJ2019211 to HW), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20191910), the Foundation for Shanghai Jiao Tong University for SMC-morning Star Youth Scholars Program, the Clinical Research Plan of SHDC (SHDC2020CR3005A), the Program of Medical Engineering Cross Research Fund of Shanghai Jiao Tong University (YG2017MS50), Clinical innovation project of Xinhua Hospital Affiliated to Medical College of Shanghai Jiaotong University (19XHCR02A), “Teaching incentive plan” project of Xinhua Hospital Affiliated to Medical College of Shanghai Jiaotong University (XH001.006.010.020), National Health and Family Planning Commission medical and health science and technology development research center key projects (NHC2018RWS01007), the Construction project of Shanghai Key Laboratory of Molecular Imaging(18DZ2260400), Shanghai Municipal Education Commission , and the Key Program of National Natural Science Foundation of China (grant 81830052).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caenorhabditis elegans (Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel ENaC/UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-α/SYD-2, Synaptobrevin/SNB-1, RAB-3, and Endophilin/UNC-57 in remodeling GABAergic neurons in  elegans . Here, wSIGNIFICANCE STATEMENT Synaptic pruning is a conserved feature of developing neural circuits but the mechanisms that dismantle the presynaptic apparatus are largely unknown. We have determined that synaptic disassembly is orchestrated by parallel-acting mechanisms that target distinct components of the active zone. Thus, our finding suggests that synaptic disassembly is not accomplished by en masse destruction but depends on mechanisms that dismantle the structure in an organized process. The nervous system is actively remodeled during development as new synapses are constructed and others are removed to refine functional circuits. Although synaptic assembly has been extensively investigated, synapse elimination is a less understood phenomenon despite its widespread occurrence . In someCaenorhabditis elegans, synapses in the GABAergic motor circuit are relocated by a stereotypical remodeling program during early larval development (A). During the first larval stage, presynaptic domains are removed from ventral DD processes and then reassembled in the dorsal nerve cord . In the resultant mature circuit, GABAergic output alternates between dorsal (DD) and ventral (VD) muscles for sinusoidal movement . Thus, these results argue that IRX-1 and UNC-8 are required for presynaptic elimination of remodeling GABAergic neurons. Additional experiments confirmed that both the Iroquois/IRX-1 and DEG/ENaC/UNC-8 normally promote the native DD remodeling program  which we have shown function with UNC-8 to promote presynaptic disassembly. Based on these findings we have proposed that UNC-8 promotes presynaptic disassembly in a pathway that depends on intracellular calcium and neural activity  were collected on a Leica TCS SP5 confocal microscope using a 63× oil objective (0.5 μm/step), spanning the focal depth of the ventral nerve cord GABA neurons and synapses. Leica application suite advanced fluorescence (LAS-AF) software was used to generate maximum intensity projections. Ventral nerve cord images were straightened using an ImageJ plug-in. Z-stack images  were collected by tracing segments of the ventral nerve cord using the segmented line tool in ImageJ (VD3–VD11). Distance in micrometers and gray value plot traces were used to count the number of peaks (synapses) that occur over the specified distance. Synapses were defined as fluorescent peaks that reached a threshold of 25 arbitrary units of fluorescence intensity. See example in Figure 3B,C.Synapse density counts ] and GFP-negative (control) VD neurons were compared with quantify differences in the fluorescence signal. Because unc-8 cDNA transgenic arrays are mosaic with expression limited to a random subset of VD neurons in each animal, data were collected from VD neurons (VD3–VD11) carrying the UNC-8 cDNA  versus an adjacent control VD neuron that does not carry the array. For results shown in Figures 7H–K, 8J,K, intensity values were obtained from line scans anterior to the VD cell bodies of interest. Background fluorescence was obtained from a line scan of an adjacent region inside the animal and subtracted from the VD line scans.FIJI was used to quantify effects of UNC-8(OE) (overexpression) on presynaptic disassembly , 8. Z-stFigure 7ic array . NeighbosmFISH was performed with custom unc-8 probes linked to Quasar 670 (Biosearch Technologies). Synchronized larvae (from either late L1 or early L3 stage) were collected by washing plates with M9, fixed in 4% paraformaldehyde in 1× PBS for 45 min and permeabilized in 70% ethanol for 48 h. Hybridization followed the manufacturer's instructions (http://www.biosearchtech.com/stellarisprotocols) and was performed at 37°C for 16 h in Stellaris RNA FISH hybridization buffer  containing unc-8 probe at 1:100. For irx-1 cell-specific RNAi , all DD motor neurons were marked with Punc-47::GFP (oxIs12) and specific DDs expressing the irx-1(csRNAi) constructs  were co-labeled with Punc-25::mCherry to distinguish them from DD neurons that did not express the irx-1(csRNAi) transgenic array. For IRX-1(OE) (IRX-1 overexpression) experiments (C), GFP-tagged IRX-1 was expressed with the ttr-39 promotor (pttr-39::IRX-1::GFP). In this setup, VDs and DDs were marked with Punc-47::mCherry (wpIs39) and individual DDs or VDs expressing IRX-1(OE) were detected by nuclear-localized IRX-1::GFP , UNC-8 was expressed in DD and VD neurons  or mCherry (C) labeled DD/VD cell bodies in both X-Y and Z axes were counted. At least 30 worms were scored for each group and the Mann–Whitney test used to determine significance (n > 45 neurons). As a positive control, unc-8 smFISH staining was noted in adjacent DA and DB ventral-cord neurons for all samples to confirm successful hybridization. csRNAi; A, all DDeriments C, GFP-taX-1::GFP . For UNCX-1::GFP H,I, UNC- juIs76; . In all  juIs76; A, 8H or  mCherry C labeledEscherichia coli. The specimens were frozen rapidly in a high-pressure freezer (Leica HPM100) at −180°C and high pressure. Freeze substitution was performed on frozen samples in a Reichert AFS machine (Leica) with 0.1% tannic acid and 2% OsO4 in anhydrous acetone. The temperature was kept at −90°C for 107 h, increased at 5°C/h to −20°C, and kept at −20°C for 14 h. The temperature was then increased by 10°C/h to 20°C. Fixed specimens were embedded in Epon resin after infiltration in 50% Epon/acetone for 4 h, 90% Epon/acetone for 18 h, and 100% Epon for 5 h. Embedded samples were incubated for 48 h at 65°C. All specimens were prepared using the same fixation procedure and labeled with anonymous tags so that the examiner was blinded to genotype. Ultrathin (40 nm) serial sections were cut using an Ultracut 6 (Leica) and collected on formvar-covered, carbon-coated copper grids . Grids were counterstained in 2% aqueous uranyl acetate for 4 min, followed by Reynolds lead citrate for 2 min. Images were obtained on a Jeol JEM-1220 transmission EM operating at 80 kV. Micrographs were collected using a Gatan digital camera at a magnification of 100,000. Images were quantified using NIH ImageJ software. Dorsal and ventral cords were distinguished by size and morphology. GABAergic synapses were identified by previously established criteria, including position in the cord as well as the morphology of the synapse. GABAergic synapses are larger than their cholinergic motor neuron counterparts, and the active zones in these synapses form a direct, perpendicular angle with muscle arms. In contrast, the presynaptic density in cholinergic synapses orient at an acute angle to the muscle, generally 30–45° and are often dyadic. Some images were collected at 30k to aid in identifying synaptic identity based on terminal position in the cord. Two colleagues with expertise in EM reconstruction of the C. elegans ventral nerve cord independently reviewed synapse images from each strain to verify identification. Each profile represents an image of a 40-nm section. A synapse was defined as a set of serial sections containing a presynaptic density with two flanking sections either side without presynaptic densities. SVs were identified as spherical, light gray structures with an average diameter of ∼30 nm. SVs were considered docked if they were in direct contact with the membrane. Three to five animals were imaged for each genotype. Numbers of profiles for each genotype were : wild type = 80/1330, unc-55; unc-8 = 37/745, unc-55;irx-1(csRNAi) = 54/613 for ventral GABAergic synapse evaluation.Young adult hermaphrodites of each strain were prepared for high-pressure freeze (HPF) fixation as described . A totalC. elegans dissection and electrophysiological methods were as previously described . The intracellular solution consisted of 120 mm KCl, 4 mm KOH, 4 mm MgCl2, 5 mmN-tris[hydroxymethyl] methyl-2-aminoethane-sulfonic acid, 0.25 mm CaCl2, 4 mm Na2ATP, 36 mm sucrose, and 5 mm EGTA . GABAergic miniIPSCs and hyperosmotic responses were acquired at a holding potential of −60 mV by pressure-ejecting extracellular saline containing an additional 500 mOsm of sucrose; 10 mm d-tubocurare (dTBC) was added to both the extracellular solution and the pressure ejection pipette to block cholinergic hyperosmotic currents. Data were acquired using Pulse software (HEKA) on a Dell computer. Subsequent analysis and graphing were performed using Pulsefit (HEKA), Mini analysis (Synaptosoft Inc.) and Igor Pro (Wavemetrics).The escribed . Animalspunc-25 GABA promoter and a C-terminal GFP tag. The resulting plasmid, pTWM90, was injected into unc-13 (e51) null mutants at 25 ng/µl with the co-injection marker pmyo-2::mCherry (2 ng/µl). This transgenic array was integrated by x-ray irradiation and outcrossed for three generations to generate stable transgenic lines for analysis.We used the In-Fusion cloning kit (Takara) to amplify the cDNA of the long isoform of UNC-13 (UNC-13L) from a plasmid provided by J. Kaplan (pTWM88). This fragment was ligated into a vector containing the pflp-13 DD promoter. The resulting plasmid, pACC98, was injected at 25 ng/µl with the co-injection marker pmyo-2::mCherry (2 ng/µl) generating the array wdEx1127. This array was later crossed with rab-3(ox785), elks-1(wy1162), and elks-1(wy1162) unc-8(syb3726) to activate GFP-tagging in DD neurons.We used the In-Fusion cloning kit (Takara) to amplify the flippase sequence from a plasmid provided by the Jorgensen lab pMLS262 (Addgene #73718). This fragment was ligated into a vector containing the unc-8(syb3726), in wy1162 [elks-1 FLP-ON GFP] animals. unc-8(syb3726) removes 6977 nucleotides from the R13A1.4b.1 unc-8 transcript except for the first 8 base-pairs and the last 44 nucleotides and thus is a likely null allele.Sunybiotech used CRISPR/Cas9 to generate a deletion allele, irx-1 RNA or containing the RNAi empty vector were seeded on NGM plates and stored at 4°C for up to one week. Four L4 unc-55, unc-55; eri-1, or unc-55; unc-8 animals were grown on each single RNAi plate at 23°C until progeny reached the L4 stage. Progeny were picked to fresh RNAi plates and the ventral synapses were quantified.Bacteria producing either double-stranded Animals were first tapped on the tail to ensure that they were capable of forward locomotion, then tapped on the head to assess ability to execute backward locomotion. Animals were binned into the following categories: unc , initiate backing (initiate backwards movement but stop), and wild type (sustain backward locomotion with at least two body bends). In irx-1(csRNAi) array (wdEx959) was outcrossed from NC2975  evaluated.For samples that are not normally distributed, the Mann–Whitney test was used to compare two groups and determine significance. For comparisons between three or more groups, we used the Kruskal–Wallis test with multiple comparison. For samples that are normally distributed, Student's D,E; unc-8 transcripts in remodeling (control) DD neurons  and detected significantly fewer unc-8 transcripts in comparison to untreated (control) DD neurons . These results are consistent with the idea that Iroquois/IRX-1 is required for DEG/ENaC/UNC-8 expression in DD neurons.In previous work, we used gene expression profiling and an RNAi screen to identify protein-encoding genes that promote presynaptic disassembly in remodeling GABAergic neurons . These sFig. 1 neurons . We notereported . We then neurons A,B. Thesirx-1 and unc-8 are not expressed in wild-type VD neurons which normally do not remodel. However, forced expression of Iroquois/IRX-1 in VD motor neurons is sufficient to trigger VD remodeling and drive the elimination of VD presynaptic terminals (irx-1(OE)] could also induce unc-8 expression in VD neurons. smFISH quantification confirmed that unc-8 transcripts are elevated in irx-1(OE) VD neurons in comparison to controls . Together, these results demonstrate that the transcription factor, Iroquois/IRX-1, is both necessary and sufficient for DEG/ENaC/UNC-8 expression in remodeling GABA neurons (E).GFP reporters for erminals . Thus, wcontrols C,D. Toge neurons E.In addition to promoting the removal of Synaptobrevin/SNB-1::GFP from ventral presynaptic domains in remodeling GABAergic neurons , UNC-8 aunc-55 mutants in which the VD GABAergic presynaptic domains are eliminated because of ectopic activation of the native DD remodeling program  . This result indicates that UNC-8 function is required for the efficient removal of presynaptic RAB-3 in remodeling GABAergic neurons . Additional experiments showed that ablation of either unc-8 or irx-1 also blocks the removal of SYD-2::GFP  in the ventral nerve cord of unc-55 mutants. Since Iroquois/IRX-1 induces UNC-8 expression (G).If Iroquois/IRX-1 activates UNC-8 expression as predicted by our smFISH results , then Ir program ; Fig. 1CFig. 1C) , 2. In t program . For exa mutants A. This r neurons . As an u mutants A–D. AddiD-2::GFP E,F in thpression , these rpression G.irx-1 encodes a transcription factor, we reasoned that Iroquois/IRX-1 might also regulate other targets in addition to the unc-8 gene in the GABA neuron synaptic remodeling pathway. If Iroquois/IRX-1 regulates a downstream target that functions in tandem with UNC-8, then genetic ablation of irx-1 should enhance the retention of ventral presynaptic markers in unc-55; unc-8 double mutants. For this test, we used feeding RNAi for global knock-down of irx-1 because the irx-1 null allele is lethal (unc-8(tm5052) deletion allele used for these experiments is a likely null mutation  in unc-55; unc-8 double mutant animals. Together, these results suggest that Iroquois/IRX-1 drives an additional genetic pathway, independent of UNC-8, that also eliminates presynaptic terminals in remodeling GABAergic neurons (D). In the next series of experiments, we used a combination of ultrastructural analysis, electrophysiology and genetics to confirm that Iroquois/IRX-1 functions in tandem with unc-8 to dismantle the presynaptic apparatus.Because s lethal . The uncmutation . We counP puncta A–C in un neurons D. In theunc-55 mutants as predicted from experiments showing that presynaptic markers  are eliminated  samples (A), thus, confirming that IRX-1 is necessary for the removal of presynaptic domains in remodeling GABA neurons.We previously used EM to establish that GABAergic presynaptic terminals are removed in iminated ; Fig. 3. neurons . Since o neurons , 4, we u samples A, thus, unc-55; unc-8 and unc-55; irx-1(csRNAi) animals are strikingly similar to wild-type GABAergic presynaptic domains (A) with normal numbers of docked SVs . Previous work has shown that SV docking at the presynaptic active zone depends on the vesicular GTPase protein RAB-3 and the RAB-3-Interacting Molecule, RIM1/UNC-10  animals  animals (I). Surprisingly, unlike other presynaptic markers, Rim1/UNC-10::GFP does not remodel in unc-55 mutants and is retained on the ventral side (I). Together, these results suggest that dual localization of both RAB-3 and Rim1/UNC-10 in ventral GABAergic synapses of unc-55; unc-8 and unc-55; irx-1(csRNAi) mutants could account for our EM observation of numerous docked vesicles . In addition, the ventral synapses detected in unc-55; unc-8 and unc-55; irx-1(csRNAi) animals show comparable dense projections (D). This characteristic presynaptic structure has been previously shown to depend on SYD-2  GABAergic synapses is consistent with the persistence of ventral SYD-2::GFP in these animals . Taken together, our EM data are consistent with the independent findings that SNB-1, UNC-57, RAB-3 and SYD-2 puncta  animals and thus support the hypothesis that functional UNC-8 and IRX-1 proteins are required for the removal of the ventral presynaptic apparatus in remodeling GABAergic neurons motor neurons.GABAergic synapses in  domains A with nocked SVs B,C. Prev1/UNC-10 . RAB-3 imodeling , but is Fig. 3A; . We addi animals I. Surpriral side I. Togethvesicles B,C. In ajections D. This con SYD-2  and its 2 puncta  are retaunc-55; unc-8 and unc-55; irx-1(csRNAi) (A–D), we next performed experiments to determine whether these synapses are functional. We first examined whether the docked SVs in unc-55; unc-8 animals are fusion-competent by recording iPSCs from ventral muscles. For these experiments, we used dTBC to block cholinergic signaling. As previously reported, tonic release of ventral iPSCs was restored in unc-55; irx-1(csRNAi) animals, but iPSCs were not detected in unc-55; unc-8 mutants despite the presence of organized clusters of fluorescent presynaptic proteins . Hyperosmotic treatment of unc-55; unc-8 animals failed to trigger ventral GABA release suggesting that these animals are defective in SV priming . As expected, hyperosmotic stimulation triggered robust iPSCs in wild-type and in unc-55; irx-1(csRNAi) animals, whereas ventral muscles in unc-55 mutants were unresponsive. To rule out the possibility of a general defect in SV fusion, we confirmed that preparations of unc-55 and unc-55; unc-8 mutants exhibited spontaneous cholinergic activity that could be abolished by dTBC . Together with our EM results  animals can dock and are also fusion competent since endogenous miniature IPSCs were detected and hyperosmotic treatment evoked robust IPSCs in unc-55; irx-1(csRNAi) adults . Thus, our results are consistent with the idea that Iroquois/IRX-1 and UNC-8 eliminate a shared set of presynaptic proteins  in the remodeling program but that Iroquois/IRX-1 selectively removes specific presynaptic components for vesicle priming and neurotransmitter release in a mechanism that does not require UNC-8.Because ventral GABAergic synapses with apparently normal ultrastructure are observed in (csRNAi) A–D, we nproteins , electro animals , thus exunc-55 mutants and reassembled in the dorsal nerve cord (D). The resultant imbalance of excess inhibitory GABAergic output to dorsal muscles versus excess excitatory cholinergic input to ventral muscles results in a striking behavioral phenotype in which unc-55 animals coil ventrally when tapped on the head instead of initiating coordinated backward locomotion (irx-1(csRNAi) restores fusion-competent GABAergic presynaptic densities to unc-55 mutants . If these restored synapses are functional, then the tapping assay should detect improved backward locomotion. Indeed, unc-55; irx-1(csRNAi) animals (unc-55 mutants (unc-55 mutants by RNAi knock-down of irx-1 (F–H). In contrast, unc-55; unc-8 mutant animals show severely defective backward locomotion that is not significantly different from that of unc-55 mutants  and reinforces the idea that ventral GABAergic synapses in unc-55; unc-8 mutants are not functional. Genetic ablation of unc-8 activity in unc-55; irx-1(csRNAi) does not further enhance backward locomotion  and by our finding that Iroquois/IRX-1 regulates expression of the unc-8 gene  animals (A), GABAergic release is selectively reactivated by knock-down of irx-1, but not by genetic removal of unc-8 (F-H). This striking difference suggests that Iroquois/IRX-1 must drive the removal of key determinants of presynaptic neurotransmitter release that are not targeted by UNC-8.As an additional test of our ultrastructural and electrophysiological results, we devised a behavioral assay to evaluate the functionality of GABAergic synapses in remodeling defective mutants . Ventralrve cord D. The recomotion . We have mutants G,H. If t animals  show rob mutants . This reof irx-1 F–H. In c mutants . This fi mutants G,H and rcomotion  as predinctional F,G and bc-8 gene . To summ animals A, GABAerof unc-8 F-H. ThisC. elegans. Because SVs appear docked, but are incapable of fusion in unc-55; unc-8 mutants , we hypothesized that UNC-13 could be absent from ventral GABAergic synapses in these animals. To test this idea, we generated a strain expressing GFP-tagged UNC-13L protein in GABA neurons. We selected UNC-13L for this experiment because it co-localizes with UNC-10/RIM whereas the short isoform, UNC-13S, shows a diffuse distribution in GABAergic motor neurons (data not shown), as also reported for C. elegans cholinergic motor neurons . This finding indicates that UNC-13L::GFP remodels to DD presynaptic domains in the dorsal nerve cord and is also a component of ventral VD synapses in the adult. We quantified the number of UNC-13L::GFP puncta in the ventral nerve cord and determined that UNC-13L::GFP is largely removed in unc-55 mutants  thus demonstrating that UNC-13L::GFP is disassembled from the ventral presynaptic domains of DD neuron and also VD neurons that undergo remodeling in unc-55 mutants. In contrast to other presynaptic markers , UNC-13L::GFP is also eliminated from ventral GABAergic synapses in unc-55; unc-8 mutants . Thus, wild-type UNC-8 activity is not required for the removal of UNC-13L from remodeling GABAergic synapses. This finding suggests that although the ventral presynaptic active zone in unc-55; unc-8 mutant GABAergic neurons appears normal by EM .The cytosolic protein Munc13/UNC-13 functions as a conserved component of the presynaptic apparatus to mediate SV fusion . Mammali mutants A–D, F–H, neurons . As prev neurons , UNC-13L neurons A,B. This mutants D,G thus  mutants E,G. ThusH,I), but does not displace UNC-13L::GFP . Together, these results show that UNC-8 function is neither necessary nor sufficient for UNC-13L removal from remodeling GABAergic synapses.Since UNC-8 expression in VD neurons was shown to drive elimination of SNB-1::GFP , we deviFig. 7irx-1 in unc-55 mutants is sufficient to restore ventral GABAergic synaptic release (F–H). Thus, we next asked whether UNC-13L::GFP is retained in the ventral nerve cord of unc-55; irx-1(csRNAi) animals. Indeed, we found that ventral UNC-13L::GFP puncta are detectable in both wild-type and in unc-55; irx-1(csRNAi) animals , thus indicating that Iroquois/IRX-1 is required for the removal of UNC-13L from remodeling GABAergic synapses .RNAi knock-down of  release F–H. Thus animals F,G, thussynapses L,M.Drosophila ELKS homolog, Bruchpilot, recruits UNC-13L/Unc13A (A). Ventral ELKS-1::tdTomato-labeled puncta are largely absent in unc-55 mutants indicating that ELKS-1 is dismantled from the presynaptic domains of remodeling GABAergic motor neurons . Ventral ELKS-1 puncta are also missing in unc-55; unc-8 mutants . Thus, UNC-8 function is not required to remove ELKS-1 from the presynaptic domain. Surprisingly, the ventral ELKS-1 signal is reduced in unc-55; unc-8 animals relative to unc-55. However, the significance of this finding is unclear because we do not observe a similar negative effect of the unc-8 mutation on ELKS-1 levels in remodeled DD neurons (D).Based on previous work demonstrating that ELKS-1 recruits the mammalian protein bMunc-13–2 to active zones and that L/Unc13A , we nextL/Unc13A  in wild-L/Unc13A A. Ventra neurons C,G. Vent mutants D,G. Thus neurons D.unc-55; unc-8 mutants , a substantial number of ELKS-1 puncta are retained in the ventral nerve cord of unc-55 animals treated with irx-1 RNAi . This finding suggests that IRX-1/Iroquois promotes the removal of ELKS-1 in remodeling GABAergic neurons but that UNC-8 is not required. For a direct test of this model, we used a transgenic strategy to overexpress UNC-8 in VD neurons . Although UNC-8(OE) in VD neurons drives the removal of ventral SNB-1::GFP , we observed that UNC-8(OE) is not sufficient to eliminate ELKS-1 . Thus, our results support the idea that Iroquois/IRX-1 removes ELKS-1 from ventral synapses of remodeling GABAergic motor neurons in a genetic pathway that is independent of UNC-8 .In contrast to results obtained from  mutants D,G, a sux-1 RNAi F,G. This neurons H,I. AlthB-1::GFP H,I, we oe ELKS-1 J,K. Thusof UNC-8 L,M.To summarize, our results show that IRX-1 drives the removal of multiple components of the presynaptic apparatus in remodeling GABAergic neurons including Synaptobrevin/SNB-1, RAB-3, liprin-α/SYD-2, Endophilin/UNC-57, UNC-13L, and ELKS-1 , 4, 7, 8unc-55 mutant VD neurons to show that both IRX-1 and UNC-8 act to dismantle the presynaptic apparatus. In addition, our findings suggest that IRX-1 functions as a transcription factor to orchestrate the overall mechanism by activating expression of UNC-8 as well as another downstream pathway that functions in parallel to drive synapse removal (L). To determine whether a similar mechanism also drives synapse elimination in the native DD remodeling program, we used an endogenous GFP::RAB-3 marker that is specifically expressed in DD neurons (A). As expected, in the wild-type, endogenous RAB-3::GFP remodels from ventral to dorsal locations during early larval development; few ventral GFP::RAB-3 puncta are detectable by the L4 larval stage (B). In contrast, unc-8 mutants at the L4 stage show significant retention of GFP::RAB-3 signal (C), likely resulting from failed synapse elimination (irx-1 in DD neurons prevents the elimination of GFP::RAB-3 (D–F), indicating that IRX-1 is also necessary for the efficient removal of presynaptic GFP::RAB-3 in DD neurons (irx-1(csRNAi)-treated DD neurons than in unc-8 mutants (F), a result consistent with the idea that IRX-1 drives expression of both unc-8 and an additional pathway for synapse elimination. This model of parallel-acting pathways predicts that irx-1(csRNAi) should enhance the synaptic removal defect of unc-8 mutants, which we also observe . Finally, genetic ablation of unc-8 does not enhance retention of residual GFP::RAB-3 by irx-1(csRNAi) (F), a result consistent with the idea the IRX-1 is required for unc-8 expression.We have exploited the ectopic remodeling phenotype of  removal L. To det neurons A. As expal stage B. In con3 signal C, likelymination . SimilarP::RAB-3 D–F, indi neurons . Notably mutants F, a resu observe E,F. Fina(csRNAi) F, a resuE,F). Because these findings were also observed for ectopically remodeling VD neurons .To summarize, our results have confirmed that (1) IRX-1 and UNC-8 promote the elimination of RAB-3 from ventral terminals of DD neurons ; (2) IRXFig. 9 neurons , 4, we punc-55 mutants showed that IRX-1, but not UNC-8, promotes removal of a specific subset of active zone proteins, ELKS-1 and UNC-13 (A). In the wild-type, GFP::ELKS-1 is initially deposited at ventral DD synapses and then relocated to dorsal DD synapses as predicted for the DD remodeling program (B). GFP::ELKS-1 is also removed from ventral DD synapses of unc-8 mutants , suggesting that UNC-8 is not required for ELKS-1 elimination. In contrast, we observed that irx-1 knock-down by irx-1(csRNAi) antagonizes the elimination of GFP::ELKS-1 from ventral synapses of DD neurons . Thus, our results demonstrate that IRX-1 but not UNC-8 drives ELKS-1 removal in DD neurons. Because Munc13/UNC-13 is also selectively removed by IRX-1 in ectopically remodeling VD neurons (G). Our findings are notable because they show that distinct genetic pathways can dismantle the presynaptic apparatus in remodeling GABAergic neurons by targeting specific active zone components.Our results obtained from ectopic remodeling of VD neurons in d UNC-13 , 8. To dd UNC-13 A. In the program B. GFP::E mutants C,D, sugg neurons E,F. Thus neurons , we prop neurons G. Our fiC. elegans  in a homeostatic mechanism that elevates neurotransmitter release to compensate for reduced postsynaptic sensitivity  while leaving others intact (VGCCs and SYD-1) to adapt the synapse to different sensory inputs ; thus, effectively “silencing” an otherwise normal appearing synapse (C). A potentially related phenomenon of synaptic silencing has been reported in the auditory circuit of the barn owl. Juvenile owls fitted with optical prisms learn to associate auditory cues with a new imposed visual location (Our work has revealed a synaptic remodeling mechanism that disables neurotransmitter release while leaving SVs and the presynaptic density intact. We showed, for example, that proteins with essential roles in SV priming, Munc13/UNC-13 and ELKS, can be selectively removed from the presynaptic apparatus in a genetic background that preserves the ultrastructural integrity of the active zone A; thus,  synapse C. A potelocation . Adaptatlocation  which colocation . The retlocation . We thus


Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements.Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus  Enterovirus genera . Nevertheless, the high similarities of their genome sequences led the International Committee on Taxonomy of Viruses to include the species RV-A, RV-B, and RV-C in the genus Enterovirus  differ very much from the severe symptoms associated with infections by some select representatives of other g., ref. . This ing., ref. . FurtherRV infections usually take a mild course with the pertinent signs of a respiratory tract irritation, typically a runny nose, a sore throat, and sometimes a slightly increased body temperature; these symptoms usually resolve within around a week . Neverthttps://www.picornaviridae.com/sg3/enterovirus/enterovirus.htm). Attempts at immunising with mixtures of inactivated prototype strains [Protection against RV via vaccination was contemplated early on and still is up to the present day ,8—for th strains  or with  strains ,12, and  strains —more rec strains . Unfortu strains ) have noEnterovirus inhibitors see refs. [Early on, preventing or at least significantly shortening the duration of the symptoms of RV infection via inhibiting crucial steps of viral replication have been sought, and various drug candidates were developed that are directed toward attachment, entry, uncoating, RNA synthesis, proteolytic processing of the polyprotein, and viral assembly . Moree refs. ,19,20,21ee refs. , reducinee refs. , or obstee refs. , are othee refs. .Enteroviruses [Amongst the best-known RV inhibitors are multi-ring organic compounds, which, early on, were mainly isoxazole derivatives. These chemicals act via stabilising the icosahedral protein shell against the structural changes required for release of the viral genome. In some cases, they also impact on viral binding to intercellular adhesion molecule 1 (ICAM-1), the cognate receptor of the majority of RVs ,33,34,35oviruses ,50,51,52oviruses ,54,55. Foviruses ,57,58, aoviruses , in vitroviruses ,60. Thisoviruses ,62 was soviruses .Enteroviruses [As shortly summarised above, a great number of capsid stabilisers, many based upon isoxazole , have beoviruses ,66,67; ioviruses . Given tpro and 3C(D)pro into the structural and the non-structural proteins Pro makes them ideal targets for inhibitors. Of note, several cellular proteins cleaved by the viral proteinases have been identified. For example, 2Apro inactivates the translation initiation factor eIF4G, shutting off cap-dependent translation [Upon entering the cytosol of the infected cell, the viral ss(+)RNA is first translated into a ~230 kDa polyprotein that is co-translationally and autocatalytically processed by the viral proteinases 2A in ref. ). The ve the RNA  and low  the RNA . Obviousnslation ,85,86,87nslation , the mitnslation . EffortsPro by screening 100,000 compounds [pro, as well as 3Cpro, are cysteine proteinases with a trypsin-like fold [Recently, high-throughput affinity selection mass spectrometry has been used to identify small molecules binding 3C(D)ompounds . Fifty pompounds  and reviompounds ,107). Itike fold ,109 and pol and the accessory 2CATPase, involved in the synthesis of ss(−)RNA and, later in infection, of massive amounts of ss(+)RNA, have been targeted as well. They are part of the membrane-associated replication complex . Foompounds . ExampleATPase appears to be involved in threading the newly synthesised RNA into partially assembled virions. It has been known for a long time that guanidine hydrochloride inhibits the replication of several picornaviruses via acting on 2CATPase, but its toxic nature has precluded its use as a drug [ATPase is also inhibited by the less toxic (S)-fluoxetine and derivatives [ATPase and, consequently, the RNA unwinding. Again, the binding site was modelled [As discussed in more detail below, the helicase 2Cs a drug . Howeverivatives , but notivatives . Dibucaimodelled , but so In addition to the inhibitors of infection acting on the virus by mechanical aggregation, entrapment, receptor blockage, and on endosomal uptake and acidification, several inhibitors of cellular proteins are listed in Table 3. Noteworthy are those that act on functions necessary for viral replication. For example, the cellular thiol hydrolase PLA2G16 was identified in genetic screens via impairment of the viral infection. PLA2G16 is important for viral RNA transfer from endosomes into the cytosol ,19,116. Enteroviruses [The heat shock protein HSP90 was shown to be required for the correct folding of viral proteins, and its inhibition reduced the viral yield of several oviruses ,120,121,oviruses . Itraconoviruses . VP0, anoviruses ,125. Contemplating the various drug targets and the corresponding drug candidates in pro, 3Cpro, and its precursor 3CDpro into four structural and seven non-structural proteins (not counting their precursors). The RV ss(+)RNA genome is approximately 7,300 nucleotides in length. At its 5′-end, it carries a peptide (VPg) of some 23 amino acid residues covalently attached via a tyrosine phosphodiester linkage. At its 3′-end is a genome-encoded poly-(A) tail of between 50 and 200 adenosines . From thATPase protein, a component of the replication complex, in encapsidation. Protein 2CATPase interacts directly with VP3, suggesting a possible involvement in threading the RNA into the assembling capsid [ATPase was also reported to play a role in uncoating [ATPase structure and function. Filling the capsid is not considered as energy-driven RNA pumping [Complementation experiments with different coxsackievirus strains point to a role of the helicase-like 2Cg capsid ,160. Unencoating  and hostncoating . The forncoating  summaris pumping . Encapsi pumping . This su pumping ,62, are  pumping , and was pumping . Early on, it was demonstrated that viral synthesis was occurring co-translationally and co-transcriptionally, and the in vitro assembly of infectious virions from the separate components, i.e., the viral capsid proteins and the RNA, was not achieved. For poliovirus, reproduction from viral RNA in vitro was accomplished, but required the presence of cellular membranous material, apparently for creating an assembly platform for the replication complex . As poinEnteroviruses [Enteroviruses, and their density corresponding to nucleotides could be clearly recognised  in the more distantly related parechoviruses to most probably constitute multiple packaging signals ; this waoviruses ,173,174.see ref. ). Nevertsee ref. .Results of in vitro experiments on RNA release from virions suggest that the unstructured poly-(A) tail, most probably the last part of the RNA to become encapsidated, is the first to emerge from the protein shell ,177. NevWe believe it rather likely that the poly-(A) tail initiates exit, maybe via passing through one of the above-mentioned holes, with the bulk of the RNA then being released through a larger orifice created by the (reversible) swinging open of one or more pentamers; as mentioned above, particles lacking one or more pentamers were seen in cryo-EM images reconstructed without imposing icosahedral symmetry ,176.+ versus K+) might modify this ‘compactness’.The RNA inside the viral shell is compact but not pressurised like dsDNA in some bacteriophages . NeverthThe RNA genome maintains its overall globular shape even after the proteolytic removal of the capsid with proteinase K under mild conditions, as visualised with atomic force microscopy (AFM) in the case of poliovirus  and withEnteroviruses [RNA-binding compounds have been present in the virologists’ toolbox for a long time: (i) acridine orange and neutral red are well known from early studies on the uncoating of several oviruses ,187, .As pointed out above, the diffusion of small molecular mass RNA double-strand-binding dyes to the viral RNA is obviously limited by the protein capsid. However, it is also limited by the RNA itself when condensed in a spherical core. As seen in As pointed out above, the viral RNA’s folding is most likely modulated by its interactions with the inner surface of the protein shell , althougSo far, we have only discussed double-stranded RNA stretches interrupted by single-stranded regions, usually appearing as loops in folding predictions . In addiHarutyunyan and colleagues assumed that RNA exit from the shell requires at least some unfolding . They thBased upon the above crosslinking results, one might assume that the stabilisation of specific secondary structure elements of the RNA might similarly hamper its exit from the viral shell. As mentioned above, typical nucleic acid sequences that can be specifically targeted are GQs . InUpon co-incubation in a Tris-HCl buffer, the small molecule GQ binder PDS  indeed sThe Eckard Wimmer lab showed that cell transfection with RNA containing a massively recoded poliovirus P1 sequence  produces a variant with an infectivity comparable to that of the native virus, provided that the encoded amino acid sequence and the GC content were maintained ,210. ThiThe mere presence of sequences predicted to adopt a GQ conformation does not attest to their corresponding folding when packed inside the capsid or when located in the cytoplasm. Therefore, it is fundamental to experimentally assess whether the predicted GQs are definitely adopting a genuine GQ fold.From the 11 sequence stretches with a predicted propensity to form GQs in RV-A2 , two werThe effect of PDS on purified RV-A2 was then studied in PBS after co-incubation at 34 °C and 4 °C, respectively. The higher temperature strongly enhances capsid breathing, which allows low molecular mass molecules such as RiboGreen, N-acetyl-aziridine, and 8-methoxypsoralen to diffuse into the virion and interact with the viral genome ,193,214;Work by Harutyunyan et al.  showed t+, contains 3 mM K+. It is well-established that the nature of cations impacts on the stability of GQs and the folding of the RNA in general [Above, the effect of PDS on the genomic RNA was only studied in PBS that, in addition to 147 mM Na general ,216. Ind general ). When t general ).Recently, short- and long-range interactions were demonstrated for the genomic RNA of enterovirus D68  and otheThe rod-like appearance of the RNA in the presence of PDS was exclusively seen in the sodium phosphate buffer. As mentioned above, viral infectivity was modulated by the incubation of purified RV-A2 with PDS under conditions allowing for diffusion through the capsid wall (unreviewed manuscript ). In agrATPase [pro [pro [pol [In the first part of this article, we provide a short and therefore necessarily incomplete review on potential viral and cellular drug targets and their corresponding inhibitors . This inATPase , the 2Apase [pro , the 3Cppro [pro , and thepro [pol . Virus bpro [pol ,150, andpro [pol ,219, thepro [pol ,220, andpro [pol , either pro [pol , activatpro [pol , interfepro [pol , and, mopro [pol .Recently, research has focused on the therapeutic potential of impacting GQ folds in DNA in cancer, particularly directed towards telomers ; for a g