Zinc (Zn), which is an essential trace element, is involved in numerous mammalian physiological events; therefore, either a deficiency or excess of Zn impairs cellular machineries and influences physiological events, such as systemic growth, bone homeostasis, skin formation, immune responses, endocrine function, and neuronal function. Zn transporters are thought to mainly contribute to Zn homeostasis within cells and in the whole body. Recent genetic, cellular, and molecular studies of Zn transporters highlight the dynamic role of Zn as a signaling mediator linking several cellular events and signaling pathways. Dysfunction in Zn transporters causes various diseases. This review aims to provide an update of Zn transporters and Zn signaling studies and discusses the remaining questions and future directions by focusing on recent progress in determining the roles of SLC39A/ZIP family members in vivo. Zn is an essential micronutrient required for growth, development, immunity, and many other physiological processes. The total amount of Zn in the human body is 2–3 g, with ~60% in the skeletal muscle, ~30% in bone, and ~5% in both, the liver and skin, while the remaining 5% is in other tissues  1]. App. App1]. Maintenance of intracellular Zn homeostasis mainly depends on two families of Zn transporters: Zrt- and Irt-like proteins (ZIPs), also known as solute carrier family 39A (SLC39A), and Zinc transporters (ZnTs), also known as SLC30A proteins, and metallothioneins (MTs) . ZIPs arZIPs and ZnTs are involved in many cellular responses, including cytokine- and growth factor-meditated signaling, and the regulation of enzymes, receptors, and transcription factors belonging to cellular signaling pathways . NumerouIn this review, we provide the updated information related to Zn transporters, focusing on ZIP family members and their roles in Zn homeostasis, cellular functions, signal transduction, development, and human diseases. We also discuss the remaining questions by reviewing recent progress in studies of Zn transporters and Zn signaling.Zn regulates a broad range of cellular functions; therefore, the dysregulation of Zn homeostasis causes various abnormalities in mammalian models ,9,10. UnZip1-KO mice are sensitive to dietary Zn deficiency during pregnancy . M. MZip7 dnd teeth . Thus, Znd teeth ,23. ThesZip8 mRNA expression is a transcriptional target of nuclear factor (NF)-κB, and ZIP8 negatively regulates proinflammatory responses through Zn-mediated downregulation of IκB kinase (IKK) activity, thereby inhibiting NF-κB activity  signaling by enhancing Akt and Erk phosphorylation . NotablyZIP10 is mainly localized to the plasma membrane, and it functions as a cell surface Zn importer ,73. As dZIP11 is localized to the nucleus and Golgi apparatus ,76. ReceXenopus tropicalis brain tissue . I. I32]. Iproteins ,83. Thesproteins , which aZip14-KO mice with dwarf body sizes, osteopenia, and impaired skeletal growth, cellular and molecular investigations revealed that ZIP14 modulates G protein-coupled receptor-mediated cAMP-CREB signaling by suppressing basal phosphodiesterase (PDE) activity  ,96. A reIn this section, we describe updated information on the role of ZIP transporters, mainly focusing on the pathophysiology. Recently, many studies of ZIP transporters have been conducted, which have improved the understanding of their crucial involvement in physiological events and showing that the loss of their functions causes diseases. Among the ZIP members, we selected ZIP7, ZIP10, ZIP12, ZIP13, and ZIP14 as ZIP transporters, of which investigations in vivo have been remarkably and rapidly progressed, so that advanced information of these molecules are reviewed below.Zip7-KO animal model. A recent investigation demonstrated that ZIP7 is highly expressed in transit-amplifying (TA) cells and Paneth cells at the intestinal crypt ..Zip14-KOAs evident from previous and current studies on the role of Zn in the physiological and pathophysiological events described above, information that is related to the biological relevance of Zn as an essential trace element is accumulating rapidly. Recent studies involving mice and humans suggested that Zn transporters have various physiological functions in tissue development and homeostasis. As described in the sections above, some studies have revealed the relation between the functions of Zn transporters and specific diseases. Despite the current progress in our understanding of the physiological functions of Zn transporters, many questions remain regarding the role of Zn transporters in health and disease.Zn transporters are expressed in various tissues and cell types, and they are localized in distinct subcellular compartments; these proteins transport not only Zn, but also Fe, Mn, cadmium, and other trace elements into subcellular compartments, indicating that the functions of Zn transporters are complex and diverse. Thus, studies clarifying the transportation mechanisms, not only of Zn but also of other metal ions, are required to understand the relation between the homeostatic mechanisms of metal ions and various diseases. This information will improve our understanding of the wide-range of functions of Zn ion and its transporters in diverse organisms. These studies may reveal how Zn ion regulates biological functions, and provide information related to its homeostasis  116]..116].According to reports on the involvement of Zn transporters in tissue development and homeostasis, the dysfunction of Zn transporters is crucial not only to disease progression, but also to disease onset. Thus, investigating the functions of Zn transporters in tissue development and homeostasis using pluripotent stem cell lines from patients with abnormal Zn-homeostasis is important for addressing these fundamental questions regarding useful disease models. If Zn transporter dysfunctions determine the onset of diseases, modulating their function by specific compounds will provide crucial clues for the development of therapeutic strategies. In addition, no studies have examined the specific compounds that are regulating Zn transporter functions, but such compounds may have great potential for the development of novel therapeutic strategies.Finally, further intensive research effort to examine Zn transporters would reveal critical molecular mechanisms that are related to mammalian health and disease.


Non‐enzymatic oligomerization of activated ribonucleotides leads to ribonucleic acids that contain a mixture of 2′,5′‐ and 3′,5′‐linkages, and overcoming this backbone heterogeneity has long been considered a major limitation to the prebiotic emergence of RNA. Herein, we demonstrate non‐enzymatic chemistry that progressively converts 2′,5′‐linkages into 3′,5′‐linkages through iterative degradation and repair. The energetic costs of this proofreading are met by the hydrolytic turnover of a phosphate activating agent and an acylating agent. With multiple rounds of this energy‐dissipative recycling, we show that all‐3′,5′‐linked duplex RNA can emerge from a backbone heterogeneous mixture, thereby delineating a route that could have driven RNA evolution on the early earth. The prebiotic emergence of the first RNA oligomers is at the crux of the origin of life. Recent reports describe the possible abiotic production of monomeric RNA precursors from simple feedstocks,We recently reported that templated ligation of mixtures of short oligonucleotides terminating with 2′‐ and 3′‐monophosphates can be made 3′,5′‐selective by means of sequential acetylation and ligation chemistry. The selectivity derives from preferential 2′‐O‐acetylation of 3′‐monophosphate‐terminated oligomers, which can then undergo regiospecific ligation. Subsequent hydrolytic removal of the 2′‐O‐acetyl groups at the newly formed 3′,5′‐internucleotide linkages completes the process. In contrast, 2′‐monophosphate‐terminated oligomers within the starting mixture are inefficiently 3′‐O‐acetylated and instead cyclize to 2′,3′‐cyclic phosphates, which undergo slower ligation.The initial 2′,5′‐cleavage results in a 2′,3′‐cyclic phosphate terminated oligomer, and hydrolysis of this cyclic phosphate is needed to access the terminal monophosphates involved in the acetylation and ligation steps of the synthetic stage. Cyclic phosphate hydrolysis is known to favor the 3′‐monophosphate product,1 annealed to its complementary template 2, and to further hydrolyze the terminal cyclic phosphate 3 generated by the cleavage  in the presence of different Mg2+ concentrations (0–40 mm), and their progress was monitored by HPLC  was stable under these conditions, with only 1 % of degradation after 24 days .Under these conditions, both the intact oligomers and the produced fragments are expected to be mainly helical , thereby preventing any degradation of single‐stranded RNA.N‐acetylimidazole, 30 °C, 1 h) and then subjected to phosphate activation . Mass spectrometry and gel analysis  were in line with our previous observations and confirmed that preferential acetylation and ligation of 3′‐phosphate‐terminated primers took place in the presence of the template as they had in its absence.In the next stage of our proposed recycling model, the fragments produced by hydrolysis would undergo regioselective 3′,5′‐ligation by means of sequential protection and activation chemistry Figure . To veriFinally, 2′‐O‐deacetylation of the newly synthesized oligonucleotide is required to access native RNA Figure . In supp1, thereby generating the fully 3′,5′‐linked product 7. Initially, oligomers 1 and 2 were annealed and the resulting duplex was hydrolyzed in the presence of Mg2+ . Aliquots of the reaction were taken at different time points . Moreover, during the last step, deacetylation and hydrolysis occur simultaneously; further cleavage of 2′,5′‐bonds takes place and the cyclic phosphate produced from the activation of 6, or residual non‐acetylated 5, is hydrolyzed back to these monophosphates and thus recycled. In accordance with the predictions of our proposed recycling model, subjecting the mixtures to further rounds of recycling resulted in the progressive enrichment in the corrected oligonucleotide 7 at the expense of 1  should be addressed to the authors.SupplementaryClick here for additional data file.


Adenosine to Inosine (A-to-I) RNA editing is a co- or post-transcriptional mechanism that modifies genomically encoded nucleotides at the RNA level. A-to-I RNA editing is abundant in the brain, and altered editing levels have been reported in various neurological pathologies and following spinal cord injury (SCI). The prevailing concept is that the RNA editing process itself is dysregulated by brain pathologies. Here we analyzed recent RNA-seq data, and found that, except for few mammalian conserved editing sites, editing is significantly higher in neurons than in other cell populations of the brain. We studied A-to-I RNA editing in stab wound injury (SWI) and SCI models and showed that the apparent under-editing observed after injury correlates with an approximately 20% reduction in the relative density of neurons, due to cell death and immune cell infiltration that may account for the observed under-editing. Studies of neuronal and astrocyte cultures and a computational analysis of SCI RNA-seq data further supported the possibility that a reduction in neuronal density is responsible for alterations in the tissue-wide editing patterns upon injury. Thus, our data suggest that the case for a mechanistic linkage between A-to-I RNA editing and brain pathologies should be revisited. RNA editing is a co- or post-transcriptional mechanism that alters the sequence of an RNA transcript relative to the genomic sequence. The most frequent form of RNA editing is a change in which genomically encoded adenosine (A) is converted to inosine (I) in stem–loop structures within precursor mRNAs. Since inosine is interpreted as a guanosine by the cellular translation machinery, editing has the potential to recode an mRNA and thereby increase the protein repertoire1ADAR1 encodes two distinct isoforms: Expression of ADARp150 (encoding a 150-kDa protein) is induced by interferon and detected mainly in the cytoplasm, and ADARp110 (encoding a 110-kDa protein) is constitutively expressed and localized exclusively in the nucleusA-to-I editing is catalyzed by ADAR enzymes. Three ADAR gene family members have been identified in vertebrates34678Drosophila1617Alu element pairs in human19202123242627Due to the rapid progress in sequencing technologies, a huge number of editing sites have been identified in recent years: several millions in human111214Editing sites that reside within the coding sequence and result in modification of the resulting protein  are rare. Only a few dozen mammalian conserved ADAR targets have been identifiedA-to-I editing levels change dynamically across developmental stages and under varying conditions: editing was shown to be lower in early stages of development and more abundant as development progress3031323435363732Brain tissues are composed of multiple cell types40In this study, we analyzed RNA-seq data obtained from purified cell populations from the mouse brain. We quantified RNA editing in these cells using various methods and demonstrated that editing levels varied considerably across cell-types. In particular, neurons exhibited the highest editing activity among all major brain cell types. We then used two separated mice cohorts of brain injury models, stab wound injury (SWI) and SCI, to demonstrate a reduction in RNA editing following injury, and argue that the apparent under-editing is accounted for by a reduction in the neuronal density upon injury. Thus, we suggest that the possibility of a change in cell-type composition  should be taken into account while analyzing any observed change in editing level found in different brain pathologies.To profile the editing pattern across all major brain cell types, we analyzed previously reported RNA sequencing data obtained from seven mouse brain cell-types  Since most editing events take place within lineage-specific genomic repeats, we adjusted the human FLNA, CYFIP2 and the Q/R site in GRIA2) were edited more strongly in neurons than in other cell types. However, a few exceptions were observed: For example, levels of editing in COPA and COG3 were higher in glial and vascular brain cell types than in neurons. We validated the differential editing levels of GRIK2 (at the Q/R site), TMEM63B, COG3, and COPA using conventional Sanger sequencing of neurons and astrocytes isolated from embryonic and neonatal origin , were collected from each animal 2 to 3 days following injury. Hippocampus was studied as it is known to be hypersensitive to injury of the cortex. Overall, the study included 24 brain samples (8 injury and 4 control for each hippocampus and cortex tissues). We then used the microfluidics-based multiplex PCR (mmPCR) methodology for targeted resequencingFLNA Q/R, TMEM63b Q/R, CACNA1D I/M, GRIK2 Q/R, GRIK2 I/V, and BLCAP Y/C. At two sites, COPA I/V and COG3 I/V, increased editing was induced by SWI. The same trend was observed in the cortex samples, although differences did not reach statistical significance . In contrast, ADARp150 transcript level was significantly elevated following SWI both in hippocampus and cortex  in the tissue at the site of injury. To visualize the cells in proximity to the injury we used DAPI staining. In the SWI model, the densities of cells were similar on injured and non-injured sides of the brain 3 days after injury . To visuNeuN, since based on the NeuN staining intensity : Editing sites that are highly edited in neurons are under-edited upon SCI, whereas those sites that are edited at low frequency in neurons (such as COG3 and COPA) exhibit elevated levels following SCI 41Alterations of RNA editing have been reported based on analyses of pathological conditions, many of which are associated with inflammation and loss of neurons. Based on our analysis, it may be suggested that, similar to the case of brain injury, some of these editing alterations are accounted for by changes in neuronal density or other changes in cell-type composition rather than to the deregulation of RNA editing. If this is indeed the case, the evidence for a mechanistic linkage between RNA editing and these pathologies should be revisited. Further, this finding may be relevant to other molecular process, such as transcription and splicing that have been studied under various conditions at whole-tissue resolution.et al.The RNA sequencing data of purified neurons, astrocytes, microglia, endothelial and various maturation states of oligodendrocytes from mouse cortex (n = 2 for each) together with whole cortex samples (n = 3) was downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO accession number GSE52564)The quality of the sequence reads was check by FastQC quality control toolAlu-specific editing detection algorithmAlu editing index, the SINE editing index is defined as the number of ‘G’s in RNA-reads nucleotides that were aligned to genomic adenosines that reside in SINE element, divided by the total number of read-nucleotides that align to SINE adenosine positions. The higher the index, the more editing occurs in SINE elements. Even though most editing sites in SINEs are very weak, thus hard to quantify reliably, the SINE index that averages over many thousands of these sites, provides a robust measure.This pipeline was applied on brain cell types and SCI RNAseq databases. We adjusted the human We used the hyper-editing analysisA list of mammalian conserved editing sites is presented in The CEI, weighted average of the editing level in all conserved sites, was evaluated for each sample in the brain cell types and the SCI databases. The index is defined as the ratio of the number of A-to-G mismatches  to the total number of reads in these sites. We excluded the two endothelial samples from this analysis since IGFBP7 was vastly overexpressed in these cells, accounting for >70% of the total FPKM value of all host genes.ADAR genes and genes containing mammalian conserved editing sites were calculated by the RSEM toolet al.Expression levels of C57B6 mice were purchased from Harlan Laboratories. Animal handling adhered strictly to national and institutional guidelines for animal research and was approved by the Ethics Committee of the Hebrew University of Jerusalem. Cortical injury experiments were performed on mice aged 6–7 weeks. For the injury experiments mice were anesthetized with ketamine/xylazine solution (50 mg/kg ketamine 7.5 mg/kg xylazine in 0.9% NaCl solution). A sterile needle was inserted vertically into the right and left cerebral hemisphere, reaching the skull surface at a depth of 5 mm. The needle was inserted through the cranium 2 mm caudal to the bregma and 1 mm lateral to the midline. The skin incision was closed with sutures. Two and three days following injury mice where sacrificed and tissue biopsy samples 3mm by 3mm of the cortex at the injured epicenter and part of the hippocampus directly beneath the site of injury were frozen.Total RNA was extracted using Trizol (Invitrogen). From three cortex samples we consecutive isolated genomic DNA and total RNA (NORGEN kit). RNA was treated with DNaseI (Roche) and reverse transcribed into cDNA using iScript™ Advanced cDNA Synthesis Kit (Bio-Rad).Brains were fixed in 4% paraformaldehyde, following by incubation in 30% sucrose for at least 24 h. Tissue sections (15 micrometers thick) were incubated for 1 h in a blocking solution consisting of 0.3% Triton X-100, and 5% goat serum. This solution was used for the dilution of both primary  and secondary antibodies. Sections were incubated in the primary antibody overnight (16 h). Slides were then washed three times (5 min each) with PBS and visualized with Cy3-labeled secondary antibody and coverslipped. At least seven sections were analyzed for each mouse.Day 2 Mouse primary cortical astrocytes were cultured as described previouslySee ADARp110, ADARp150, ADARB1, ADARB2, NeuN and CD45 was determined using qRT-PCR on the 7900HT Fast Real-Time PCR System . For each tested gene, we used 14 injured samples and 7 or 8 biological control samples (from cortex and hippocampus origin). cDNA sample were tested in triplicates using the PerfeCTa SYBR Green FastMix  using the following specific primers:The relative mRNA quantification of ADAR-p110: 5′-GCAGCGTCCGAGGAATCG-3′ and 5′-TAAGACTCCGGCCCCTGTG-3′ADAR-p150: 5′-CACTATGTCTCAAGGGTTCAGGG-3′ and 5′-CACTTGCTATGCTCATGACTAGGG-3′ADARB1: 5′-GTCCTGCAGTGACAAGATAGCAC-3′ and 5′-GGTTCCACGAAAATGCTGAG-3′ADARB2: 5′-GGGGAGCAGCTAATCACCAT-3′ and 5′-TGGAGGTACACAGGCTCGAT-3′NeuN: 5′-GGTGGAGTTGCTGGTTGTCT-3′ and 5′-GCACAGACTCATCCTGAGCA-3′CD45: 5′-TAGGCTTAGGCGTTTCTGGA-3′ and 5′-TCGTGCCCAAACAAATTACA-3′The relative quantification of gene expression levels were normalized to CHMP2A expression level (NM_014453) using the following gene specific primers; 5′-AAGGCCAGATGGATGCTGT-3′ and 5′-CCGCATCAACACAAACTTGC-3′ and the ΔΔCt method was appliedTo confirm the editing level obtained by RNAseq and targeted resequencing, PCR was performed using Phusion Hot Strart II High-Fidelity DNA Polymerase (Thermo Scientific) to amplify FLNA, TMEM63B, GRIK2 Q/R, COPA and COG3. The PCR program was set according to manufacture recommendation. Resulting PCR amplicons were purified using PCR Purification kit (Invitrogen) and editing was determined using the reverse primer by Sanger sequencing (Applied Biosystems).We used the SINE editing value together with editing levels of conserved editing sites, followed by pairwise correlation and clustering performed by gplot package in R, to compare the editome of the various cell-types. As the data showed a clear separation between neurons and non-neurons (R > 0.64 without neurons while R > −0.09 when including neurons), we chose the astrocyte as a representative non-neuron cell type, calculated the differences between editing levels in neuron and astrocyte samples, and correlated them (Pearson) to the differential editing signal observed for SCI sample vs. control.Animal handling adhered strictly to national and institutional guidelines for animal research and was approved by the Ethics Committee of the Hebrew University of Jerusalem.How to cite this article: Gal-Mark, N. et al. Abnormalities in A-to-I RNA editing patterns in CNS injuries correlate with dynamic changes in cell type composition. Sci. Rep.7, 43421; doi: 10.1038/srep43421 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases.Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded  Through transcription and translation, disease proteins carrying an extended polyQ stretch are detected in the affected tissues. It is widely accepted that polyQ disease toxicity is ascribed to the misfolding and aggregation of disease proteins4. However, an accumulation of evidence demonstrates that the expanded CAG-repeat RNA also contributes to toxicity in polyQ diseases8. Various expanded CAG RNA pathogenic pathways have recently been described, including the recruitment of muscleblind-like (MBNL) proteins to expanded CAG-repeat RNA foci10, the generation of small CAG RNAs via Dicer cleavage12, and the activation of nucleolar stress14.Polyglutamine (PolyQ) diseases, including Machado-Joseph Disease (MJD) and Huntington’s disease, are a group of dominantly inherited progressive neurodegenerative diseases characterized by the existence of expanded CAG trinucleotide repeats within the coding region of disease genesCAG RNA triggers nucleolar stress and eventually induces toxicity both in vitro and in vivo14. Nucleolar stress is a cellular response to the failure in ribosome biogenesis and/or ribosome malfunction15. A reduction in ribosomal RNA (rRNA) transcription causes an imbalance in the intracellular levels of ribosomal RNAs and ribosomal proteins, which triggers ribosome assembly defects and eventually leads to nucleolar stress-induced apoptosis17. Our previous investigation showed that expanded CAG RNA physically interacts with the nucleolin (NCL) protein13, a multifunctional nucleolar protein that plays critical roles in precursor rRNA (pre-rRNA) transcription18, processing19 and pre-ribosome assembly20. This RNA-protein interaction leads to upstream control element (UCE) hypermethylation and the down-regulation of rRNA transcription, which induces nucleolar stress13. We developed a 13-amino acid peptide inhibitor, P3, which can inhibit NCL-expanded CAG RNA interaction and suppress RNA toxicity in polyQ diseases21. The P3 peptide was designed based on the structure of the RRM2 domain of NCL21 which contains the ribonucleoprotein domain-1 (RNP-1) motif22. Our study showed that P3 could directly and preferentially bind to expanded continuous CAG-repeat RNA in vitro21. This P3-CAG RNA interaction titrated endogenous NCL away from binding to the toxic RNA and restored NCL-UCE interaction and pre-45s rRNA transcription21. Treatment of P3 suppressed expanded CAG RNA-induced cell death in a mammalian cell model and neurodegeneration in a Drosophila disease model21. The calculated maximal inhibitory concentration (IC50) of P3 in inhibiting cell death was 4.369 ± 1.140 μM21. In this study, we engineered peptide P3 and identified a more potent inhibitor for targeting RNA toxicity in polyQ diseases.We previously demonstrated that expanded 21. We previously synthesized a series of P3 analogues, in which basic or aromatic residues were substituted with Ala and their binding to expanded CAG RNA was measured to identify the amino acid side chains involved in the interaction with expanded CAG RNA. Our results showed that Lys3, Lys5, Tyr9, and Phe12 are indispensable and serve as the pharmacophores for RNA binding21. To further illustrate how the mutations affect the interaction between P3 and RNA, we performed isothermal titration calorimetry (ITC) experiments to characterize the interaction between each individual mutant  and entropy , suggesting that the peptide-RNA interaction is energetically driven by both favorable enthalpic and entropic components.P3 is derived from the primary structure of NCLD of P3MT3 and P3MT4 revealed that the mutations of the aromatic residues reduced the binding affinity to 16 and 17 μM respectively, whereas mutations of the lysines  had a more dramatic effect and reduced the affinity by ~3–12 fold  and Trp (P3V4) also improved the binding of peptide to expanded CAG RNA by nearly 2- to 4-fold, respectively. These results indicate that although alterations of the size or polarity of the aromatic residues improved the binding affinity of P3, only modest improvements were obtained. On the other hand, the KD value of P3V5, in which Lys3 and Lys5 both mutated to Arg residues, was approximately 0.86 µM, reflecting a nearly 10-fold improvement over the P3 peptide. Contrarily, when we mutated all three Lys to Arg (P3V6), the KD of the peptide increased to 23 µM, indicating that the replacement of the amine of Lys13 with a guanidinium is not favored.To improve the binding affinity and specificity of P3 toward expanded rg Table . SubstitD value of P3V7 was determined to be 2 µM using ITC, indicating that C-terminal amidation improved the peptide affinity to expanded RNA. When the N-terminus was also acetylated, the dissociation constant of P3V8 was further lowered to 0.33 µM , expanded CAG RNA (MJDCAG78 RNA), and expanded CAA/G RNA (MJDCAA/G78 RNA), respectively, to investigate whether the binding of P3V8 is RNA expansion-dependent and sequence-dependent  synthesis occurs within the nucleolus15. We previously showed that expanded CAG RNA induces nucleolar stress by preventing the nucleolar protein NCL from binding to the upstream control element of the rRNA promoter, leading to the downregulation of rRNA transcription13. To investigate whether P3V8 suppressed neurotoxicity by alleviating expanded CAG RNA-induced nucleolar stress in vivo, we measured the expression levels of pre-rRNA in DsRedCAG100 flies treated with the peptide. Real-time PCR analysis showed that P3V8 could restore the transcript level of pre-rRNA in a dose-dependent manner. When DsRedCAG100 flies were treated with 1 μM of P3V8, the pre-rRNA level was fully recovered to the DsRedCAG0 control level  to deliver P3WT and P3V8 to EGFPCAG78 RNA-expressing HEK293 cells. Figure CAG RNA-induced cell death. Although P3V8 alone did not exert any cellular effect, its administration with the aid of DX could effectively alleviate the cytotoxicity induced by expanded CAG RNA  and integrated time-concentration responses  of L1P3V8 were also significantly higher than that of P3V8  and P3V8 (15 pmol/g) in the brain were achieved 20 min after intranasal dosing, as compared to those from intravenous administration, indicating better brain uptake via the intranasal route  rats were studied and compared Fig. . The maxV8 Table , suggestute Fig. . Taken tCAG RNA induces cytotoxicity, we recently developed a peptide inhibitor P3 that disrupts NCL-RNA interaction and consequently mitigates RNA toxicity in polyQ diseases. In this study, we determined the equilibrium dissociation constant KD of P3 using ITC and discovered that the binding of P3 to RNA is both enthalpically and entropically favored. Short peptides like the 13-mer P3 usually assume flexible conformations in an aqueous solution and therefore suffer entropic loss upon binding to their targets. The finding that P3 binds expanded RNA with a favorable TΔS suggests that P3 may have undergone a conformation change upon binding to the RNA in solution.Despite our growing knowledge of the role of RNA transcripts in the pathological mechanisms of polyQ diseases, there is a lack of inhibitors that can counteract their neurotoxicity. On the basis that sequestration of NCL by expanded CAG RNA, we found that the basic Lys residues are more important than the aromatic residues in the interaction with expanded RNA. Substitution of any of the three Lys in P3 greatly reduced its binding affinity to RNA, indicating that electrostatic interactions play a dominant role in the affinity of P3. This is no surprise because CAG-repeat RNA duplex adopts an A’ helical conformation that is intermediate to the A- and B-forms of nucleic acids and is highly electronegative on its surface and thus charge-complementary with the key pharmacophores of P3. As described above, the substitution of Lys at positions 3 and 5 by Arg remarkably improved the binding affinity of the peptide by nearly 10-fold (Table When characterizing the effects of Ala substitutions of the pharmacophores of P3 on its interaction with expanded CAG RNA (KD =0.33 µM) but also significantly improved its potency both in vivo and in vitro. In a recent study of compounds that block Alzheimer’s Aβ channel activity, Flora and colleagues showed that capping of the amine and carboxyl groups of free histidine helps to improve the residue activity by preventing nonspecific interaction with other reactive residues in the target28. We speculate that the capping of P3, now termed P3V8, might have exerted a similar effect and prevented nonspecific interaction between the peptide and the RNA’s negatively charged surface, thus improving the binding and efficacy of the peptide. Furthermore, the slight increase in pI of the peptide might also have contributed to its better affinity to the electrostatically negative surface of RNA .Previous studies have shown that the charge neutralization of the N- and C-termini of synthetic peptides by acetylation and amidation can improve their stabilities in cell cultures and serum, thus improving their biological activity. Based on these findings, we generated P3V8, which is capped by acetylation and amidation at the N- and C-termini, respectively, in an attempt to improve the biological activity of P3 and in hopes that the same strategy can be applied to other P3 derivatives. We observed that capping of both termini of P3 not only improved its binding affinity to expanded D to about 2.3 µM. We speculate that upon N- and C-termini modifications, the interactions of the arginines and RNA might have been altered and that P3V9 likely adopts a different binding mode than P3V5 or P3V8. Moreover, the lengths of the basic side chains at residues 3 and 5 also appear to play a determining role in the binding of the peptide, as either lengthening or shortening the basic side chains abolished the interaction between the inhibitors and expanded RNA, suggesting that the distances between the basic side chains and the RNA is critical and that only limited conformational flexibility is allowed after P3V8 binds to the RNA. Such a rigid binding mode of P3V8 is further supported by the observation that tandem repeats of the peptide failed to interact with the expanded RNA. Studies on the conformation and dynamics of P3V8 before and after RNA binding are needed to understand the mechanism of its specific inhibitory activity.With the success of P3V8, we applied the same capping strategy to P3V5 to further improve its activity. However, as described above, the combination of Lys-to-Arg mutations of residues 3 and 5 and the capping of the peptide termini did not improve the peptide’s binding affinity but instead reduced the KCAG RNA is the widened major groove caused by the non-canonical AA base pairs29. Such widening provides binding sites that are unique to CAG-repeat RNA, and their accessibility may offer opportunity to improve the potency and specificity of our inhibitor. Further structural information on the interaction between P3V8 and CAG RNA will be useful to provide insight into how to make use of such a unique feature. Nevertheless, the results of our study demonstrates that neutralization of the N- and C-termini by simple modifications like acetylation and amidation can have significant effects on the binding and inhibitory properties of peptidyl inhibitor against an RNA target.RNAs are becoming more recognized as attractive therapeutic targets because they fold into well-defined secondary and tertiary structures but exhibit a large variety of conformations, which can provide favorable opportunities for specific drug targeting. One interesting feature of the A’ form of 33 or CAG RNA toxicity34 in polyQ disease have been developed. Some even showed therapeutic potential33. In this study, we demonstrated for the first time a structural activity relationship (SAR) investigation of the peptidylic inhibitor, P3, toward CAG RNA toxicity in polyQ diseases. The SAR study led us to identify a more potent peptide derivative of P3, P3V8, which shows dramatically improved inhibitory efficacy against expanded RNA-mediated toxicity in vivo. However, many peptides have short half-life in vivo, typically in minutes, which raises concern whether P3V8 could be administered as a therapeutic agent. Previous studies have established that covalent anchoring of lipids to peptides promotes peptide binding to the fatty acid binding sites on albumin35. As albumin has a very long half-life, this ‘docking’ into albumin can significantly extend the functional half-life of peptides. Furthermore, we speculated that lipidation of a peptide could improve the ability to cross cell membranes and enter cells. We therefore lipidated P3V8 by N-acylation with palmitic acid. Stability studies of the lipidated peptide L1P3V8 in plasma and brain homogenates revealed that lipidation significantly enhanced the cellular uptake and in vivo stability of P3V8. Above all, the lipidation strategy notably improved the pharmacokinetic profile and brain uptake of L1P3V8 in rats when it was administered intranasally, conferring our inhibitor drug/lead-like properties. The ability of therapeutic agents to pass the blood-brain-barrier is one of the most critical requirements for treatment of neuronal diseases. Although the mechanism of brain uptake of L1P3V8 remains to be elucidated, our results illustrate that the combination of lipidation and intranasal administration may provide a new means to improve the brain uptake of therapeutic agents. In conclusion, our results provide proof of concept that lipidated peptidyl inhibitors that target RNA toxicity are a novel therapeutic option for polyQ diseases. Further modification and optimization on this lead-like L1P3V8 will be needed to prolong its half-life and uptake level in the brain.In the past few decades, several inhibitors targeting protein toxicity23. The sequence of the 13 amino acid peptide was submitted to the server and 100 simulations were performed using the default settings. The program returned the most representation conformations identified in terms of energy and population, and clustered them based on their sOPEP  coarse grained energies. The representative model of the top-ranked cluster with the lowest sOPEP energy values was selected for this study.Molecular modeling of P3V8 was carried out using the online PEP-FOLD 2.0 serverCAG27, pcDNA3.1-MJDCAG78, pcDNA3.1-MJDCAA/G78 and pEGFPCAG78 constructs were reported previously13.The pcDNA3.1-MJDtert-butyloxycarbonyl . Amino acids, NMP, DMF and piperidine were supplied by Iris Biotech (Germany). Acetonitrile, formic acid, triethylsilane (TES), trifluoroacetic acid (TFA) acetic anhydride, and dichloromethane (DCM) were from Sigma-Aldrich (Denmark). All chemicals were used as received and without further purification.P3WT and P3 mutant (MT) peptides were purchased from GenScript USA Inc. All other peptide variants were prepared by Fmoc solid-phase peptide synthesis on automated peptide synthesizers, Biotage Syro Wave and Biotage SP Wave instruments, and assembled on a 0.1 mmol scale, unless noted otherwise. Peptide syntheses were carried out on a TentaGel S Rink Amide resin (0.22 mmol/g). Amino acids had Fmoc protection of Nα-amino groups; side-chain protecting groups were tert-butyl , 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl , and 3CN-H2O (0.1% formic acid), connected to an ESI-MS . Purification of the peptide was performed on a preparative Dionex Ultimate 3000 HPLC with a C18 column Phenomenex Gemini Axia . Unless otherwise stated CH3CN-H2O (0.1% TFA or 0.1% formic acid) was used as eluent with a flow of 15 mL/min. Gradient elution for 0–5 min was 5%, then to 55% for 5–32 min.Analytical HPLC was performed with a Dionex Ultimate 3000 instrument on a Phenomenex Germini-NX C18 column , column oven thermostated to 42 °C, and a linear gradient flow of CHAll standard Fmoc amino acids were coupled in DMF with 5.2 equivalents of amino acids and HOAt, with 5 equivalents of HBTU and 9.4 equivalents DIEA. The coupling time was 60 min at room temperature. All non-standard amino acids, as well as palmitic acid, were coupled using 2 equivalents of amino acids and HOAt, 1.9 equivalents of HBTU and 3.6 equivalents DIEA. Coupling times were 10 min at 75 °C. N-terminal fatty acid was introduced using the same conditions as for the coupling of standard amino acids.2O (95:2:3 or 95:2.5:2.5) for 2 hr. The TFA solutions were concentrated under a flow of nitrogen and the compounds were precipitated with diethylether to yield the crude products. All peptides were purified by RP-HPLC.In the synthesis of peptides P3V7 to P3V21, the first 10 couplings used a coupling time of 60 min at 25 °C with NMP and washings in-between couplings. Deprotections were performed by treatment with piperidine-DMF (2:3) for 3 min, followed by piperidine-DMF (1:4) for 15 min. After each coupling and deprotection, a washing procedure with NMP (3x), DCM (1x), then NMP (3x) was performed. From the 10th coupling onwards, the coupling time was increased to 2 × 120 min and an extra deprotection step (15 min) with piperidine-DMF (1:4) was added. The N-terminal acetylation was achieved with acetic anhydride in DMF (1:4) for 2 × 15 min. After completion of peptide chain assembly, the resin was washed 6 times with DCM. All the peptides were released and deprotected by treatment with a cocktail of trifluoroacetic acid (TFA), triethylsilane (TES) and Hin vitro binding was over 90%. Desalted peptides were used in Drosophila feeding assays. All RNAs were synthesized using the MEGAscript® kit (Ambion) as previously described13, and the MJDCAG27, MJDCAG78 and MJDCAA/G78 RNAs were transcribed from linearized pcDNA3.1-MJDCAG constructs36.The sequences of peptides are listed in Table ® scientific plotting software version 7 . All RNAs and peptides were dissolved in binding buffer  and 0.7 mM of peptide was titrated into 0.5 µM of RNA for each experiment. The concentration of RNA was estimated with appropriate extinction coefficients at 260 nm on a Nanodrop 2000 (Thermo Scientific). A reference power of 8 μcal/s was used with an initial 0.5 µl of injection of peptide followed by 2 µl for all subsequent titrations points with a 60 sec initial equilibrium delay and 150 sec pause between injections. The samples were stirred at a speed of 1000 rpm throughout the experiment. The thermal titration data were fitted to the ‘one binding site model’ to determine the dissociation constant (KD). Each experiment was repeated at least 3 times with consistent results obtained.Experiments were carried out using a MicroCal iTC200 isothermal titration calorimeter  at 25 °C. Data were analyzed using the Origin2 in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Primary rat cortical neurons were isolated and cultured as previously described37. Transient transfection of HEK293 cells was performed using Lipofectamine 2000 (Life Technologies). P3V8 were delivered to HEK293 cells using the DeliverX (DX) Peptide Transfection Kit (Affymetrix) 4 hr after DNA transfection. At least two batches of independently synthesized peptides were used in the experiments.HEK293 cells were cultured at 37 °C with 5% COEGFPCAG78 RNA-expressing HEK293 cells, a LDH assay was employed. HEK 293 cells were seeded on a 24-well plate at a density of 0.5 × 105, and pEGFPCAG78 DNA construct was used to transfect the cells. For P3V8 treatment, different concentrations of P3V8 as indicated in the figure were transfected by DeliverX transfectant 4 hr after DNA transfection. For L1P3V8 treatment, different concentrations of L1P3V8 were added into individual wells immediately after DNA transfection. Seventy-two hours after treatment, LDH enzyme activity in the cell culture medium was measured as described before. Experimental groups were normalized to the untransfected control. After normalization, data were analyzed using the dose response-inhibition curve (nonlinear regression-variable slope) to determine the IC50 value . Each experiment was repeated at least 3 times.To detect the effect of P3V8 and lipidated P3V8 (L1P3V8) on inhibiting cell death in UAS-DsRedCAG0 and UAS-DsRedCAG100 were kind gifts of Professor Nancy Bonini . The gmr-GAL4 fly line was obtained from Bloomington Drosophila Stock Center. For pseudopupil assay, third instar larvae were fed with various amount of respective peptides dissolved in 2% sucrose solution for 2 hr and then continued to culture in standard fly food at 21.5 °C38. Pseudopupil assay was performed on 12 day-old adult flies as mentioned previously39. Images were captured by SPOT Insight CCD camera controlled by the SPOT Advanced software (Diagnostic instruments Inc.). Image processing was performed using the Adobe Photoshop CS software (Adobe). Each experiment was repeated at least 3 times (n = 10 fly heads), and consistent results were obtained. For viability test of wild-type flies from egg to adult stage, eggs born within 5 hr were collected and cultured in fly food containing 10 or 50 μM of respective peptides at 21.5°C. Viability from egg-to-adult was calculated as the number of adult flies collected divided by the number of eggs examined. Each experiment was repeated for three times (at least 130 eggs were examined in each group). Two batches of independently synthesized peptides were used in the experiments.Flies were raised at 21.5 °C on cornmeal medium supplemented with dry yeast. Fly lines bearing 13. The following probes were used: pre-45s rRNA (Assay ID: AILJIZM), pre-rRNA (Assay ID: AIMSG5U), Drosophila GAPDH (Assay ID: Dm01841186) and human actin (Assay ID: Hs99999903_m1). Each experiment was repeated at least 3 times.RNA was extracted from cells or ten 12 day-old adult fly heads by Trizol reagent (Life Technologies), and 1 μg of purified RNA was then used for reverse-transcription using the ImPromII™ Reverse Transcription System (Promega). Random hexamer (Roche) was used as primers in reverse transcription. Taqman gene expression assays were performed on an ABI 7500 Real-time PCR system and data were analyzed as previously describedSprague-Dawley (SD) rats  were supplied by the Laboratory Animal Services Centre at The Chinese University of Hong Kong. All animal studies were conducted under the approval of the Animal Ethics Committee of The Chinese University of Hong Kong, and were performed in accordance with relevant guidelines and regulations. Blank rat brain homogenate was prepared by ultrasonic probe homogenization  of brains collected from the control rats. Blank rat plasma was prepared by centrifugation (8000 rcf for 3.5 min) of blood collected from control rats. For the stability test, 2000 ng/mL and 500 ng/mL of P3V8 or L1P3V8 was spiked into the blank rat plasma or brain homogenate and vortexed. The mixtures were incubated at 37 °C with 100 rpm in a water bath for 1 or 3 hr. The incubation was terminated by addition twice the volume of acetonitrile and centrifugation at 13000 rcf for 10 min. The supernatant was collected for LC/MS/MS analysis.5 and cultured overnight at 37 °C with 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were then treated with 100, 500 or 1000 nM of respective peptide at 37 °C for 3 hr. After treatment, the cells were washed with ice-cold PBS and then lysed by 2% SDS solution. Aliquot (100 µl) of the cell lysate was added with 200 µl acetonitrile and centrifuged at 13000 rcf for 10 min to precipitate the proteins. The collected supernatant was subjected to LC/MS analysis. The protein concentration of the cell lysate was measured using a bicinchoninic acid protein assay kit following Sigma’s protocol.HEK293 cells were seeded on a 6-well plate at a density of 6 × 10SD rats (180–200 g) were anesthetized with an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (6 mg/kg) and received a minor surgery of cannulation with a polythene tube  in the left jugular vein. The rats received an overnight recovery with free access to food and water. In the following day, rats were administered P3V8 or L1P3V8 (3 μmol/kg) via intravenous or intranasal routes. For intravenous administration, appropriate volume of P3V8 or L1P3V8  was injected to the rats via the cannula. For intranasal administration, the rats were temporarily anesthetized by inhalation of carbon dioxide, and was administrated with appropriate volume of P3V8 or L1P3V8  in both nostrils by a micropipette. Chitosan solution  was pre-treated to rats intranasally 5 min before the P3V8 and L1P3V8 was administrated via intranasal routes. For the pharmacokinetic study, after the drug administration, blood samples were collected from the catheter at appropriate time intervals . After each collection, 0.2 mL of saline containing 25 IU heparin was injected to compensate for the blood loss. Plasma was collected after centrifugation at 8000 rcf for 3.5 min and stored at −80°C until analysis. For brain uptake study, at 20 min, the rat was anesthetized and perfused by 500 mL saline, and the whole brain was collected. The brain was wiped by tissue paper to remove excess water, meninges and blood vessels followed by storage at −80 °C until analysis.Aliquot of plasma sample (80 μL) was mixed with 160 μL acetonitrile. The mixture was vortexed for 1 min and followed by centrifugation at 13000 rcf for 10 min. The supernatant was collect and subjected to LC/MS/MS analysis.To prepare brain homogenate, the whole brain of each rat was minced. Saline (2 mL/g brain) was added followed by ultrasonic probe homogenization on ice. For analysis of P3V8, aliquot of brain homogenate (100 μL) was mixed with 200 μL acetonitrile. The mixture was vortexed for 1 min and followed by centrifugation at 13000 rcf for 10 min to collect the supernatant. For analysis of L1P3V8, aliquot of brain homogenate (650 μL) was centrifuged at 6000 rcf for 10 min. After the supernatant was collected, 400 μL saline was added to re-suspend the pellet. The mixture was again vortexed for 5 min and centrifuged at 6000 rcf for 10 min. The supernatant collected was loaded to a preconditioned Oasis® HLB cartridges . After washed with 1 mL of 10% acetonitrile in water, the analyte was eluted with 0.25 mL 95% acetonitrile in water.Agilent 6430 Triple Quadrupole LC/MS/MS system  was employed for the analysis. For analysis of P3V8, chromatographic separation was achieved on a SunFire C8 Column . The mobile phase of water containing 0.2% formic acid (A) and acetonitrile (B) was used with a gradient elution . The flow rate was 0.8 mL/min. Multiple reactions monitoring (MRM) with fragmentation transition of 500 to 129 in positive ion mode was employed for quantization of P3V8. For analysis of L1P3V8, Alltima Amino Alltech Column  was used for separation. Mobile phase of water (A) and acetonitrile (B) was used. The gradient elution for plasma samples was 0–4 min, 30–95% B, and for brain samples was 0–9 min, 10–95% B. The flow rate was 0.8 mL/min. MRM transition of 565 to 88 in positive ion mode was used for quantization of L1P3V8.post hoc Tukey test or unpaired t-test. “*”, “**”, “***” and “****” represent P < 0.05, P < 0.01, P < 0.001 and P < 0.0001 respectively, which are considered statistically significant.Plasma concentration verse time profiles were analyzed by WinNonlin  to obtain the pharmacokinetics parameters. Other data were analyzed by one-way ANOVA followed by Supplementary information


KIF1A has been shown to be important for experience dependent neuroplasticity. In Drosophila, amorphic mutations in the KIF1A homolog unc-104 disrupt the formation of mature boutons. Disease associated KIF1A mutations have been associated with motor and sensory dysfunctions as well as non-syndromic intellectual disability in humans. A hypomorphic mutation in the forkhead-associated domain of Unc-104, unc-104bris, impairs active zone maturation resulting in an increased fraction of post-synaptic glutamate receptor fields that lack the active zone scaffolding protein Bruchpilot. Here, we show that the unc-104brismutation causes defects in synaptic transmission as manifested by reduced amplitude of both evoked and miniature excitatory junctional potentials. Structural defects observed in the postsynaptic compartment of mutant NMJs include reduced glutamate receptor field size, and altered glutamate receptor composition. In addition, we observed marked loss of postsynaptic scaffolding proteins and reduced complexity of the sub-synaptic reticulum, which could be rescued by pre- but not postsynaptic expression of unc-104. Our results highlight the importance of kinesin-3 based axonal transport in synaptic transmission and provide novel insights into the role of Unc-104 in synapse maturation.The kinesin-3 family member  C. elegans Unc-1044Drosophila Unc-104/Imac7KIF1A knockout mice display severe neurological abnormalities including motor and sensory disturbances and die shortly after birthDrosophila complete loss of unc-104 function in motor neurons leads to an arrest of synaptogenesis and embryonic lethalityKinesins are microtubule based molecular motors that transport various cargos including membranous organelles, protein complexes and messenger RNAs2KIF1A have been associated with hereditary spastic paraplegia (HSP)9101112KIF1B has been implicated in Charcot-Marie-Tooth diseaseKIF1A were also described to affect the central nervous system causing mental retardation and brain atrophy916unc-104’s mammalian homolog KIF1A is important in facilitating synaptogenesis in an experience dependent mannerDrosophila photoreceptorsKIF1A function.In humans recessive, autosomal dominant and spontaneous mutations in , unc-104bris which causes partial loss of kinesin-3 function and thus permits detailed analysis of synapse maturation at the larval stage unlike the unc-104 null mutant that dies at late-embryonic stageunc-104bris at the forkhead associated (FHA) domain causes morphological changes at the neuromuscular junction (NMJ) such as increased NMJ length and synaptic bouton number as well as reduced bouton sizeunc-104brishave increased proportion of postsynaptic glutamate receptor fields that are unapposed by presynaptic active zone (AZ) scaffolding protein Bruchpilot (Brp)202122unc-104 in presynaptic maturationWe have previously reported a hypomorphic alleleunc-104 loss-of-function on the maturation of the presynaptic terminal, little is known about its impact on postsynaptic development. In the present study, we examine functional and structural defects at unc-104bris mutant NMJs with emphasis on structural deficits in the postsynaptic compartment. We found defects in synaptic transmission and altered glutamate receptor composition as a consequence of partial loss of kinesin-3 function. In addition, the postsynaptic compartments are characterized by the reduced abundance of discs large (Dlg), Dorsal B (DorB) and α-Spectrin (α-Spec) as well as reduced folding complexity of the subsynaptic reticulum (SSR). These defects can be rescued by the presynaptic but not postsynaptic expression of unc-104. Collectively, our data reveal new functions of kinesin-3 based transport in synapse development and may have important implications for our understanding of the molecular pathway underlying neurological diseases caused by impaired kinesin-3 function.Despite the mounting evidence on the consequences of Unc-104bris mutant larvae demonstrate a distinct synapse maturation defect. At wild-type NMJs only the youngest synapses, in sum approximately 5%, lack the AZ organizing protein Brpunc-104bris mutant larvae about 25% of the postsynaptic densities (PSDs) are unapposed by Brp20rd instar larval NMJs. The amplitude of evoked and miniature excitatory junctional potentials (EJPs and mEJPs) was reduced by 72% and 50% respectively in unc-104bris mutants compared to controls . We observed a 17% reduction of the average GluR field size at the NMJ  including PSD protein 93 and 95 , SAP97 and SAP102 are thought to be key organizers of molecular scaffolds at excitatory synapsesant NMJs . Next, wunc-104bris could be a result of loss of Unc-104 in the presynaptic or postsynaptic compartment or both. Thus, we ectopically expressed UAS-unc-104-mcherry in the unc-104bris background either pan-neuronally using elav-Gal4 or in muscles using 24B-Gal4. Presynaptic but not postsynaptic expression of Unc-104 was able to restore normal Dlg abundance in mutant NMJs  membrane trafficking4748unc-104bris mutant NMJs  unc-104bris larvae is similar to those described for a Drosophila model for SPG10 caused by dominant negative mutations in kinesin heavy chain (khc), the Drosophila homolog of human KIF5Aunc-104bris larvae suffer from a more generalized paralysis, i.e. NMJs in segments innervated by longer axons seem not to be more severely affectedThe degree of behavioral impairment and the lethality of KIF1A has been associated with another neurological disease HSAN2CKIF1A have been associated with SPG30 as well as non-syndromic intellectual disability accompanied by variable additional symptoms including progressive encephalopathy and brain atrophy9161753While truncation of unc-104bris larvae has been suggested to further stabilize kinesin-3 dimers by electrostatic interaction with the E499 residueunc-104bris larvae are not restricted to the axonal compartment, but include defects in dendrite maturation in sensory neuronsThe amino acid mutated in w1118, elav-Gal4, 24B-Gal4 and unc-104d11024 were obtained from the Bloomington Drosophila Stock Center. GluRIIA-mRFPunc-104-mcherry was a generous gift from Thomas Schwarz (Harvard University).Flies were cultured on standard soft media seeded with live yeast at 25 °C unless otherwise indicated. rd instar stage larvae were dissected in Ca2+-free HL3 solution and fixed in 4% formaldehyde in PBS for 3 minutes (for staining with native fluorescent proteins) or for 10 minutes (for staining with only immunofluorescent labeling). Correct NMJs were identified as previously described202357Middle 3Sample preparation and image acquisition were performed essentially as previously describedrd instar stage larvae as previously described2, 10 NaHCO3, 5 trehalose, 115 sucrose, and 5 HEPES; the concentration of Ca2+ was 1, pH adjusted to 7.2. All experiments were performed at 18 °C. Stimulation of the segmental nerve was executed by pulling the cut end of the nerve into a self-made suction electrode (5–6 μm in diameter) filled with HL3 and passing a brief (0.3 ms) bi-polarizing pulse across the nerve. Stimulation was accomplished with an ISO-STIM 01D stimulus isolation unit . The signal was acquired with an Axoclamp 900A amplifier (Axon Instruments), digitized with a Digidata 1440A analog to digital board, and recorded with a PC using pClamp 10. 3 (Axon Instruments), and analyzed with AxoGraph X software. The amplitudes of the EJPs were corrected for nonlinear summation. The quantal content was estimated by dividing the averaged corrected EJP by the averaged mEJP amplitudeCurrent clamp intracellular recordings were performed on muscle 6 segment A2 of mid 3p < 0.05; **p < 0.01; ***p < 0.001. Data were first tested for normality and then analyzed by either the student’s t-test for two groups or by a one-way analysis of variance followed by a Tukey’s multiple comparison test. Non-normally distributed data were analyzed by using either a Mann-Whitney test for two groups or a Kruskal-Wallis test for multiple groups.Statistical analysis was performed using the software PRISM 6. Sample errors are given as standard error of the mean (SEM). The following alpha levels were used for all tests: *This study did not involve the use of human subjects or samples from human donors. All experiments were performed in accordance with University guidelines and regulations.How to cite this article: Zhang, Y. V. et al. The KIF1A homolog Unc-104 is important for spontaneous release, postsynaptic density maturation and perisynaptic scaffold organization. Sci. Rep.7, 38172; doi: 10.1038/srep38172 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Staphylococcus aureus expresses a set of transcriptional factors and small RNAs (sRNAs) to adapt to environmental variations. Recent harmonization of staphylococcal sRNA data allowed us to search for novel sRNAs using DETR’PROK, a computational pipeline for identifying sRNA in prokaryotes. We performed RNA-Seq on Newman strain and identified a set of 48 sRNA candidates. To avoid bioinformatic artefacts, we applied a series of cut-offs and tested experimentally each selected intergenic region. This narrowed the field to 24 expressed sRNAs, of which 21 were new and designated with Srn identifiers. Further examination of these loci revealed that one exhibited an unusual condensed sRNA cluster of about 650 nucleotides. We determined the transcriptional start sites within this region and demonstrated the presence of three contiguous sRNA genes  expressed from the positive strand, and two others (srn_9343 and srn_9346) transcribed from the opposite one. Using comparative genomics, we showed that genetic organization of the srn_9342-9346 locus is specific to Newman and that its expression is growth-phase dependent and subjected to nutrient deprivation and oxidative stress. Finally, we demonstrated that srn_9343 encodes a secreted peptide that could belong to a novel S. aureus toxin-antitoxin system.The human pathogen  Staphylococcus aureus is an opportunistic pathogen responsible for a large spectrum of human and animal infections1. The bacterium finely modulates gene expression to efficiently adapt its growth and physiology to the local environment. Besides global transcriptional regulators, small RNAs  have emerged as key players in a wide range of biological processes, from central metabolism to virulence and antibiotic resistance4. In S. aureus, sRNAs, are typically 50- to 500-nucleotides (nt) long. Some of them regulate mRNA expression and/or stability without the need of Hfq chaperone6. sRNAs with regulatory functions were first discovered by Mizuno in E. coli7 and then by Novick in S. aureus8. They can modulate target mRNA expression by cis- or trans-acting mechanisms2. Cis-encoded sRNAs are transcribed from the opposite strand of an mRNA or another sRNA. Accordingly, they display perfect complementarity with their target sequences, although their ability to bind other RNAs cannot be excluded. Trans-encoded sRNAs are transcribed apart from their targets, and usually display only partial complementarity with them.S. aureus sRNAs via bioinformatics, next-generation sequencing (NGS), and other experimental approaches21. This has resulted in the publication of hundreds of RNA sequences, but their functions are mostly unknown. Most of these studies were done using methicillin-resistant strain N31518. In others, newly discovered sRNAs were described based on their genomic location in that particular strain16. S. aureus sRNA identification has been hampered by the lack of naming consensus and the absence of dedicated annotation files. Recently, we collected all of the sequences published so far, proposed a simple sRNA gene identifier (srn) to avoid redundancies, and provided annotation files in the Gene File Format (.gff) to the SRD (Staphylococcus regulatory RNA Database)22. In this study, the sRNA content of four strains was compared, and their predicted locations suggested that sRNA content is strain-dependent. It therefore seems that global analysis devoted to the identification of novel sRNA is still useful for many bacteria, including S. aureus. This was recently confirmed by studies conducted on multilocus sequence typing 8 (ST8) strains, with new sRNAs described in USA300 and NCTC832521. Here, we combined bioinformatics and experimental procedures to identify and characterize novel sRNAs expressed by the S. aureus strain Newman23. This is a methicillin-susceptible clinical isolate often used to study staphylococcal diseases in animal models23, and it belongs to the common ST8 clonal lineage. However, Newman has never before been used to discover new staphylococcal sRNAs, and only three previous studies have focused on the role of already identified sRNAs in Newman. The first described the Srn3580_SprA type I toxin-antitoxin system24; the second focused on the anti-virulent role of Srn_3610_SprC25; and the third highlighted the functional role of Srn3820_SprX1 in Newman pathogenicity26.Over the last decade, several studies have focused on the identification of Δsrn_3610_sprc. We combined the RNA-Seq data with the initial sRNA annotation from the SRD22, then used the DETR’PROK pipeline27 to identify 48 putative novel transcribed intergenic regions (IGRs). Bioinformatic and experimental analysis of the 48 loci led to the certain characterization of more than 20 novel sRNAs expressed from either the core or accessory genome. Further study of these sRNAs along with RACE-mapping of primary transcripts revealed that Newman has an unusual condensed cluster of five sRNAs. These are all located within 650 nucleotides, and their expression is growth-phase dependent and subject to nutrient starvation and oxidative stress. In the cluster, two RNA pairs are expressed as sense/cis-antisense sRNA, with one pair presenting toxin-antitoxin module features, since one of the sRNA was demonstrated to encode and express a 33 amino acid long secreted peptide. This work provides evidence for the existence of at least 21 new S. aureus sRNAs, and highlights the unprecedented organization of an sRNA cluster of unknown function. Our approach could be extended to wider experimental conditions on the most represented STs to characterize the full repertoire of staphylococcal sRNA (pan-RNome).In this study, we performed deep RNA sequencing on Newman wild-type and its isogenic mutant 22. The majority were identified using high-throughput screening, with sixty experimentally confirmed22. Our goal here was to search for novel sRNAs by combining RNA-Seq, bioinformatics, and experimental assessments, thus reducing the false discovery rate inherent to this type of approach. We examined S. aureus Newman, a methicillin-susceptible clinical isolate29 which has never before been used for sRNA discovery. Whereas previous studies were based on the use of various growth conditions21, we compared wild-type Newman to an sRNA mutant strain, as sRNAs can modulate the expression of various targets including transcription factors 3. These transcription factors could, in turn, control sRNA expression. Therefore, we used a strain deleted for Srn_3610_SprC sRNA to see whether that deletion allowed the identification of more sRNAs. Srn_3610_SprC is an sRNA that belongs to the SarA regulon and attenuates virulence30. Indeed, in the absence of Srn_3610_SprC, S. aureus phagocytosis by macrophages increases, allowing for an internal proliferation of the bacterium and its subsequent release and dissemination into the organism. During the post-exponential phase, we extracted total RNA from isogenic and mutant strains, and prepared RNA-Seq libraries. We then used DETR’PROK27, a pipeline recently developed to identify sRNAs in prokaryotes. DETR’PROK assembled, in the non-annotated regions, overlapping reads into clusters representing sRNA candidates. Consequently, the workflow was designed and set to preferentially search for RNAs transcribed in an independent manner rather than mRNA leaders (see Methods for the parameters used). To avoid reexamining previously reported sRNAs, we combined the SRD gene annotation for srns22 with gene-coding annotations in GFF format that were downloaded from NCBI. Among the six biological replicates (three wild-type Newman and three Newman Δsrn_3610_sprc transcriptomes), there were 18 to 27 novel sRNA candidates per replicate, resulting in a total of 48 independent IGRs that were not from mRNA UTRs  transcript level variations for any of the IGRs. Consequently, it indicated that none of these 48 clusters were under the control of Srn_3610_SprC. This result can be explained by recent work showing this sRNA to be directly repressed by SarA which therefore significantly lower expression of Srn_3610_SprC in wild-type under normal growth condition30.So far, more than 500 sRNAs are compiled under unique Srn identifiers in the SRDRs Table . To veri22. To address this issue, each sequence extracted from DETR’PROK’s output was systematically submitted to BLAST to determine whether the candidate sequence appeared elsewhere in the Newman genome. From the 48 IGRs listed in Table 31 lower than 20. Third, FPKM (fragments per kilobase of exon per million reads mapped) normalization33 was done to take into account not only the sequencing depths, but also the lengths of the transcribed regions. We eliminated all the clusters with an average FPKM of less than 9. The combination of these cutoffs led to the removal of 23 clusters  located respectively in φNM1 and φNM2 are almost identical, with more than 98% sequence identity over 380 nt , a 2- to 4-fold increase in RNA levels was observed at an OD600nm of 6, followed by a decrease   was used as an internal control for northern blot analysis monitoring the expression of the 17 IGRs during growth in brain-heart infusion (BHI) medium at four time points Fig. . In thiste) Fig. . The trate) Fig. . For thete) Fig.  exhibitete) Fig. . To asseted Fig. . This suS. aureus reference strains. To do that, we ran a BLAST query to look for homologous IGR sequences in the S. aureus N315, HG003, USA300, and UAMS1 genomes39. Among those that northern blots showed as being expressed, only IGRs 1050586, 1079951, 2083121, and 2605426 were retrieved in each of the four genomes, and their transcription was confirmed by northern blots . Two by two, they share the same loci but are transcribed from opposite strands. This made us wonder whether the remaining 13 transcripts were associated with transcription located on the opposite strand and undetected by DETR’PROK due to the average library size and the sequencing mode (2 × 100 bp). To investigate this, and because so far the S. aureus RNAs transcribed from the opposite strand of a known sRNA are usually smaller than 100 nt22, we performed a new RNA-Seq. In this one, we made and purified cDNAs with a minimal length of about 70 bp and generated single reads of 50 nt (sequenced using MiSeq), while the size of the smallest fragments generated for HiSeq sequencing was larger than 100 bp. We then used Artemis to specifically look for reads mapping on the opposite strand of these 13 RNAs, finding five additional read clusters  and Srn_9660 (IGR_2017461), were transcribed. We also used TEX followed by polyphosphatase treatment to identify a TSS for srn_3765 (IGR_2083121) at position 2083472 (a 276-nt transcript), while an RNA fragment of only 204 nt was detected without TEX treatment, suggesting maturation of that particular RNA at position 72  were already identified as transcribed in those two recent studies  was not demonstrated. We also used the Uniprot database to search for sequence homology with known proteins or domains. Eight of them did not match any referenced proteins or peptides, while the two remaining shared partial homologies with larger proteins. Together, these data indicate that 14 of the identified transcripts identified are bona fide non-coding sRNAs, and that the features of the remaining 10, make them good candidates for sRNA with potential dual-functions. Overall, it shows that the S. aureus sRNA content has not been fully uncovered yet.We previously compiled staphylococcal sRNAs into the SRD database under simple srn identifiers followed with their alignment showed that some sRNAs (mostly those starting with srn_9XXX) were not detected in the four SRD reference strains . Therefore, to estimate the conservation levels of the 24 novel sRNAs, we aligned their sequences to assess their presence and absence in 22 S. aureus strains that belong to well-characterized clonal complexes  are conserved among several S. aureus strains and have high nucleotide sequence identities , while other strains contain none or just two or three of these sRNA genes  and srn_9345/srn_9346, whereas a three-CDS insertion occurred where Newman has srn_9343/srn_9344. In CA-347, srn_9342-srn_9344 organization is conserved, but the last two sRNA genes are lacking. Interestingly, Mu50 and JH1 have two copies of srn_9342 , and an organization similar to that of NCTC8325 is also present elsewhere in the genome. In JH1, three loci contain sRNA genes of Newman’s srn_9342-srn_9346 locus, with srn_9342 again found in two copies (the first at the 3′ end of an autolysin amidase gene). The srn_9345/srn_9346 gene cluster displays an organization that resembles that of Mu50. In a third genomic environment, srn_9342 and srn_9343/srn_9344 are located next to another autolysin amidase domain, upstream from an HtrA-like protein coding-gene. Therefore, the comparative analysis showed that the genetic organization of the accessory srn_9342-9346 sRNA genes is specific to Newman, and that differences in the nearby genetic environment may induce differences in sRNA expression patterns.The analysis of these new sRNAs in various srn_9343/srn_9344 revealed that their presence is less conserved than that of the other genes in the locus. While no ORF was identified on the positive strand of the srn_9342-9346 locus, analysis of the minus strand revealed a small ORF within the srn_9343 sequence . Western blot analysis did not reveal any translation of the fusion peptide in cell extracts or in the Newman supernatant  and Peptostreptococcaceae bacterium . Altogether, the presence of transcription on both strands, as for Srn_3580_SprA24, and similarity with the RelE toxin suggests that srn_9343/9344 could belong to a putative toxin-antitoxin system.Analysis of the distribution of ce Table . It contges Fig. , with a ant Fig. , indicatrns Fig. , indicatrns Fig.  and secr48. Therefore, the expression of the 11 srns detected by northern blot was assessed under various conditions, including some that mimic the changes encountered by S. aureus during host infection. Cells were grown in TSB until reaching the exponential phase of growth (OD600nm = 2), then subjected to stresses related to pH , temperature (heat and cold shocks), oxidation, osmotic levels, and nutrient availability. After either 30 or 60 minutes of stress, the sRNA levels were monitored by northern blot and compared with those produced under unstressed conditions , and remained high in Srn_2335 after one hour of stress. Nutrient deprivation (NZM) in both Srn_3765 and Srn_9348 led to decreased RNA levels after both 30 minutes and 1 hour. Similarly, Srn_9680 levels were around 3-fold lower after one hour in NZM medium. Cold shock (18 °C) in Srn_3765 led to increased RNA levels , whereas Srn_2335 level was multiplied by six after 1 hour. Finally, heat shock (42 °C) led only to an induction of Srn_5075. Interestingly, the two copies (Srn_9650 and Srn_9660) as well as Srn_9670 and Srn_9680 (located near Srn_9660) respond to the same stresses, suggesting similar regulatory mechanisms.Many sRNAs respond to specific environmental modifications and help regulate dedicated physiological networksons Fig. . Among aons Fig. . Converssrn_9342-9346 locus responds to stress similarly to srn_9344 (which is sensitive to oxidative stress and cold shock), we investigated the expression levels of these srns. We used RT-qPCR rather than northern blot as we found that Srn_9343 and Srn_9346 are expressed at low levels in RNA extracts  and facilitates the identification of new sRNAs. Recent publications have demonstrated that knowledge of the full repertoire, the pan-sRNome, has not yet been achieved21, as it varies significantly according to the strain22. S. aureus epidemiology indicates that clonal complex 8 lineages are widely distributed worldwide and dominate human carriage and infections49. We therefore continued our efforts to extend the S. aureus pan-sRNome by studying the Newman clonal complex 8 strain. This strain has been fully sequenced23 and used successfully in animal models of staphylococcal infections, but its sRNA content is quite under-investigated.The last decade has led to the discovery of most 27 to search for unannotated transcribed regions within the Newman IGRs. This resulted in the identification of 48 loci for which RNA-Seq data allowed read clustering. Although DETR’PROK identified sRNA content differences between the Newman and the Newman Δsrn_3610_sprc strains, differential expression analysis revealed no statistical differences between the two. Previous studies underlined the high probability of identifying repeated sequences, or 5′ or 3′ UTRs22. To avoid this issue, we assigned specific parameters to DETR’PROK, used several cut-offs, systematically used BLAST, and viewed the read mappings to determine whether sRNA candidates seemed to be independent transcriptional units. In addition, misannotated genes were removed during a final manual cull , and when it reaches the next rho-independent terminator the transcription stops. Analysis of the minus strand revealed the presence of an ORF within srn_9343 along with an RBS upstream the predicted AUG initiation codon. Using 3xFLAG, we showed that the Srn_9343 peptide is produced and secreted. However, failure to express this peptide in Newman suggests the existence of a specific regulatory mechanism that shuts down srn_9343 expression and translation. Looking outside the Staphylococcus genus, this peptide sequence presents similarities to the bacterial RelE toxin. RelE is a part of a type II toxin-antitoxin module55 in which the toxin RelE acts as a ribosome-dependent endoribonuclease activated in diverse cellular processes, including nutrient starvation and persistence56. The antitoxin encoded by RelB counteracts RelE’s toxic activity by modifying the structure of the RelE catalytic domain57. During amino acid starvation, RelE inhibits translation by cleaving within the mRNA ribosomal A-site. Interestingly, our expression analysis conducted in NZM-poor medium showed that Srn_9344 slightly decreased, while RNA levels of Srn_9342, Srn_9345, and Srn_9346 increased. However, no RelB homologues could be identified next to Srn_9343 or elsewhere in the Newman genome, suggesting that Srn_9343 may not be a part of a type II toxin-antitoxin system in S. aureus. The architecture of the srn_9342-9346 locus is unusual in S. aureus. The presence of an sRNA (Srn_9344) transcribed from the opposite strand of the peptide-coding Srn_9343, and at the same locus, suggests that Srn_9344 could function as a cis-antisense. We hypothesize that this pair may function as a type I toxin-antitoxin system. Apart from S. aureus, another unusual sRNA cluster was recently identified in the Enterococcus faecalis clinical isolate V583, where a chromosomal locus containing two toxin-antitoxin modules (type I and II) with cis-antisense transcription was discovered58.A careful study of the 17 transcribed IGRs began with MiSeq, which led to the discovery of five additional sRNAs. This was completed with RACE-mapping, which characterized the 24 independent transcriptional units or primary transcripts. According to Rfam and Riboswitch finder46 or any peptide/proteins with a known function. Many published sRNAs were described as having coding capacities22 and there are accumulating evidences for the existence of RNAs with dual functions in prokaryotes and eukaryotes60. In S. aureus, this is the case for multifunctional regulatory RNAIII, that besides monitoring the expression of a large set of mRNA targets in its RNA form3, encodes the delta-hemolysin. This raises the question of whether the term “noncoding RNA” sometimes used by the RNA community is still relevant and may suggest that some of the sRNAs described here may harbor dual-functions. Recent work in S. aureus showed that Srn_4340 (Teg23), which contains an ORF, is probably a small non-coding RNA rather than a small coding sequence, since no translation was seen at all21. In that study, the authors emphasized that current automatic annotation methods can lead to genome overannotation, therefore complicating the identification and study of new sRNA. It therefore appears that the distinction between the mRNA and sRNA world (apart from comparing RNA lengths) is probably more complicated and intertwined than expected, and that thorough experimental assessments of potential coding capacities and regulatory functions may be necessary.Our search for ORFs within the 24 sRNAs mapped by RACE revealed that some of them may encode peptides or small proteins, although none of them are homologous to phenol-soluble modulins22 allowed us to investigate the distribution of the 24 srns within several S. aureus strains. This search revealed that seven of them were conserved among the strains we analyzed. Aligning these sRNA sequences revealed a high level of conservation, with only few SNPs. Such high conservation is usually observed for transcribed regions and is significantly decreased in true IGRs. One of the open questions is whether there are still additional sRNAs to discover in S. aureus. Based on the work we present here and on other recent studies21, all performed on different strains in the same clonal complex (CC 8), we can reasonably suppose that the annotation of the pan-sRNome is far from being complete. Therefore, the study of other clonal complexes and using various experimental conditions should help in the identification of novel sRNAs which could be specific to clonal complexes or conserved within the species and therefore belong to the sRNA core. Investigations could be extended to the study of strains deleted for transcription factors, since it was recently shown that sRNAs could belong to regulons in S. aureus30. Indeed, studies of the expression of our novel sRNAs under various stress conditions revealed that they respond to few specific stresses, and that their expression is therefore tightly monitored. Interestingly, our data indicated that many of these novel sRNAs are downregulated under oxidative stress. This can be opposed to the observation made in N315 during the identification of the Teg sRNAs11, implying that each sRNA responds to its own set of physico-chemical modifications.The assignment of Srn identifiers as per a procedure established in SRDS. aureus RNome, and this step is essential before trying to understand its overarching role and function within the bacterium and especially during infection. Our work shows that the search for novel sRNAs in well-studied bacteria is valuable, and that this can be facilitated through setting conditions that mimic the in vivo environment, by using different RNA-Seq approaches, or by studying strains deleted for major transcription factors to start characterizing the complex connections between sRNAs and proteins in this major human pathogen.In conclusion, including the 24 novel sRNA coordinates in the SRD will enable subsequent RNA-Seq studies using various experimental conditions to monitor their physiological role. Global transcriptome approaches are necessary for an exhaustive characterization of the S. aureus strains used in this study are listed in Table S. aureus was grown in Brain Heart Infusion broth  or in Tryptone Soya Broth  under agitation at 37 °C (except where stated). Growth was monitored by measuring the OD600nm at different time points. For the analysis of RNA levels under different stresses, S. aureus was cultured in TSB broth up to an OD600nm of 2 . Stresses were induced by the addition of 10 mM H2O2 (oxidative stress), HCl (to lower the pH to 5.5), NaOH (to increase the pH to 9.5), or by temperature shifts to 18 °C or 42 °C (cold and heat shocks). In addition, 60 ml of culture was centrifuged at 4500 rpm for 8 min at room temperature and the pellet resuspended in TSB supplemented with 1 M NaCl (osmotic stress), NZM medium (stringent response), or fresh TSB as a control.The Cells were harvested by centrifugation at 4500 rpm for 10 min and pellets washed with 500 µL of cold lysis buffer . Cell pellets were broken using acid-washed glass beads (Sigma) in the presence of phenol (pH 4) in an FP120 FastPrep cell disruptor  for 30 sec at a power setting of 6.5. Lysates were centrifuged at 16,000 g for 5 min at 4 °C. Total RNA were extracted with phenol/chloroform and precipitated overnight with sodium acetate.S. aureus were diluted in fresh BHI broth to an OD600nm of 0.1 then cultured at 160 rpm for 5 hr at 37 °C. Total RNA were extracted as described above and treated with Amplification Grade DNase I (Invitrogen) to remove genomic contaminations. The absence of DNA was checked by qPCR in an Applied Biosystems 7500 instrument and RNA integrity verified on a Bioanalyzer (Agilent). Ribosomal RNAs were depleted using a Ribo-Zero Magnetic Kit (Epicentre) according to the manufacturer’s recommendations. Stranded cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). The concentration, quality, and purity of the libraries were determined using a BioAnalyzer, a Qubit fluorometer (Invitrogen), and a Nanodrop spectrophotometer (Thermo Scientific). Libraries were sequenced on either an Illumina MiSeq instrument  or an Illumina HiSeq 1500 system , as per the manufacturer’s instructions.Overnight cultures of 22 were added to this GFF. Quality control of RNA-Seq reads and read mappings was performed as previously described22. SAM files were filtered on bitwise flag values61 to select properly paired reads. Fragments sorted in SAM files were counted by HTSeq count31 for stranded library with the union mode and differential expression calculated using DESeq32. BAM files were visualized using the Artemis browser34.The Newman strain genome sequence and annotation file (in GFF format) was obtained from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Staphylococcus_aureus_Newman_uid58839/). All of the SRD srns described for Newman strain27 workflow of 43 steps to identify novel RNAs in S. aureus Newman. Briefly, the pipeline clustered overlapping RNA-Seq reads from BWA alignments to identify novel transcripts in intergenic regions using a GFF file that combined 2614 CDS; 56 tRNAs; 16 rRNAs; RNaseP RNA; 4.5 S RNA; tmRNA; and 504 SRD-listed srns. We set DETR’PROK to identify and retain clusters of more than 12 overlapping reads that were located at least 25 nt apart from existing annotations and had a length over 50 nt. DETR’PROK then produced a list of candidates in a GFF annotation file.We used the DETR’PROK48. Briefly, 15 µg of total RNA were loaded and separated in 7% polyacrylamide/8 M urea gels. To estimate the actual length of transcripts, a DynaMarker® Prestain Marker for Small RNA Plus was loaded into each gel. RNAs were probed with γ32P 5′ endlabeled oligonucleotides (File 41 with some modifications (see below), using the primers listed in File 41.Northern blots were performed as previously describeddes File  and detesrn_9343 was cloned in pCN35 under its own promoter or under Pveg promoter44 and in fusion with a 3xFLAG (GACTACAAGGACCACGACGGTGACTACAAGGACCAC GACATCGACTACAAGGACGACGACGACAAGTGA) using the primers listed in File S. aureus as previously described30. S. aureus was cultured in BHI broth at 37 °C and under agitation until reaching an OD600nm of 6. Cells were harvested by centrifugation, and cellular protein extracts were prepared with protease inhibitors as previously described41. Proteins from the supernatant were precipitated with TCA and then with acetone as previously described41. For western blots, samples were separated on 16% Tricine-SDS-PAGE and transferred onto Hybond-P PVDF membranes (Amersham). After overnight blocking at 4 °C, membranes were incubated with ANTI-FLAG horseradish peroxidase-conjugated antibodies (HRP) (Sigma). The membranes were revealed using an ECL Plus Western Blotting Detection Kit (Amersham) and scanned with an ImageQuant LAS 4000 imager.42. RNA samples  were treated with 1 unit of Amplification Grade DNase I, and the absence of DNA contamination was checked by qPCR using an Applied Biosystems 7500 system. cDNA were synthesized from total RNA (250 ng) using a High-Capacity cDNA Archive Kit (Applied Biosystems) and specific primers to discriminate the strand transcribed. Real-time PCR was done using a QuantiTect SYBR Green PCR Kit (Qiagen) in an Applied Biosystems 7500 real-time PCR system. Data were analyzed using the comparative critical threshold (ΔΔCT) method as previously described: the target RNA amounts were compared with HU or 5 S RNA, which served as internal controls25. The primers used for cDNA synthesis and qPCR are listed in File Total RNA extraction was performed as described aboveet al.22, previously registered with GEO under accession number GSE64026 were used to enhance the number of replicates.The RNA-Seq data generated during this study (by MiSeq and Hiseq) was submitted to the GEO repository under accession number GSE89487. Additionally, reads obtained from the work of Sassi Supplementary informationDataset 1Dataset 2Dataset 3Dataset 4


High-throughput genomic technology has enabled us to screen the entire genome and generate hypotheses at relatively low costs. One of the driving forces in high-throughput genomic technology is high-throughput sequencing (HTS). With its rapid development and affordability, HTS has quickly become the go-to choice for interrogating the entire genome. The analysis methodology development for HTS has been at the forefront of bioinformatics in recent years. Hundreds of tools and pipelines have been developed to aid researchers in interpreting HTS data. The aim of this special issue is to promote research and reflect the most recent advances in addressing HTS data analysis.KM Mouse Strain.” In this article, the authors studied the chromosome 1 sequence of the Chinese Kunming mouse and compared the sequence to three other mouse species.Title: “Chromosome 1 Sequence Analysis of C57BL/6J-Chr1Title: “The Utilization of Formalin Fixed-Paraffin-Embedded Specimens in High Throughput Genomic Studies.” In this review article, the authors thoroughly examined the practicability of conducting high-throughput genomic assays, including HTS using formalin-fixed paraffin-embedded specimens.Title: “An Integrating Approach for Genome-Wide Screening of MicroRNA Polymorphisms Mediated Drug Response Alterations.” In this study, the authors examined the relationship between polymorphisms and drug response in microRNA.Title: “Comparative Transcriptome Analysis Reveals Effects of Exogenous Hematin on Anthocyanin Biosynthesis during Strawberry Fruit Ripening.” Through RNA sequencing, the authors examined the expression change of genes in strawberries that had been applied with exogenous hematin.Pinctada fucata, Based on Transcriptome Analysis.” Through RNA sequencing, the authors studied changes in the gene expression pattern during the metamorphic development of a pearl oyster.Title: “Differential Gene Expression during Larval Metamorphic Development in the Pearl Oyster, Title: “RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining.” In this study, the authors examined the efficiency of two ribosomal RNA deletion kits: Ribo-Zero and RNase H.We received a total of 14 manuscripts and, through rigorous review, selected six for publication in this special issue. What follows is a brief summary of the six manuscripts:


The next-generation sequencing (NGS) technology has revolutionized our previous understanding of the plant genomes, relying on its innate advantages, such as high throughput and deep sequencing depth. In addition to the protein-coding gene loci, massive transcription signals have been detected within intergenic or intragenic regions. Most of these signals belong to non-coding ones, considering their weak protein-coding potential. Generally, these transcripts could be divided into long non-coding RNAs and small non-coding RNAs (sRNAs) based on their sequence length. In addition to the well-known microRNAs (miRNAs), many plant endogenous sRNAs were collectively referred to as small interfering RNAs. However, an increasing number of unclassified sRNA species are being discovered by NGS. The high heterogeneity of these novel sRNAs greatly hampered the mechanistic studies, especially on the clear description of their biogenesis and action pathways. Fortunately, public databases, bioinformatics softwares and NGS datasets are increasingly available for plant sRNA research. Here, by summarizing these valuable resources, we proposed a general workflow to decipher the RDR (RNA-dependent RNA polymerase)- and DCL (Dicer-like)-dependent biogenesis pathways, and the Argonaute-mediated action modes (such as target cleavages and chromatin modifications) for specific sRNA species in plants. Taken together, we hope that by summarizing a list of the public resources, this work will facilitate the plant biologists to perform classification and functional characterization of the interesting sRNA species. Ten years after the accomplishment of the first plant genome project , “miR168—AGO18” in rice (Oryza sativa), and “miR390—AGO7” in both plants , natural antisense transcript-derived small interfering RNAs (nat-siRNAs), and phased small interfering RNAs (phasiRNAs). Specifically, hc-siRNAs are encoded within the heterochromatic loci transcribed by RNA Pol IV. The single-stranded Pol IV transcripts are converted to double-stranded precursors through the RDR2 (RNA-dependent RNA polymerase 2)-dependent pathway. Then, the precursors are processed by DCL3 for the production of the 24-nt hc-siRNAs. The hc-siRNAs are incorporated into AGO4 to perform site-specific chromatin modifications  of the primary TAS transcripts are the prerequisite for initiating ta-siRNA production. Through the RDR6-dependent pathway, the cleaved TAS transcripts are converted to double-stranded precursors which will be subject to ta-siRNA processing by DCL4. Finally, most of the ta-siRNAs are loaded into AGO1 silencing complexes to guide target cleavages       Table , could aAs introduced above, there are two major forms of sRNA precursors that could be processed by DCLs, i.e. the long double-stranded RNA (dsRNA) precursors and the single-stranded RNAs with short internal double-stranded regions Fig. . The forcis- and trans-NAT pairs of 70 plant species. Gene locus ID could be used as a query to see the possibility of this gene to form NAT pairs with other genes. Optionally, “batch download” could be selected to obtain the complete list of the predicted NAT pairs of a plant species. In the other way, the researchers could perform large-scale NAT prediction by using the program NATpipe    (Willmann et al., The AGO4-associated sRNAs, such as the hc-siRNAs, repress gene expression through chromatin modifications Chen, . By usin“Target mimicry” was reported as a novel pathway for the regulation of the miRNA activities by the target mimics (Franco-Zorrilla et al., Finally, researchers could construct a sRNA-centered regulatory network involving sRNA targets and target mimics by using Cytoscape (Shannon et al., In the present work, we proposed a general workflow for deciphering the biogenesis and action pathways of the plant sRNAs by using a series of publicly available resources. Most of the recently reported sRNA-seq and dsRNA-seq datasets of Arabidopsis and rice were summarized in Table