Mononegavirales are highly diversified eukaryotic viruses including significant human pathogens, such as rabies, measles, Nipah, and Ebola. Elucidation of their unique strategies to replicate in eukaryotic cells is crucial to aid in developing anti-NNS RNA viral agents. Over the past 40 years, vesicular stomatitis virus (VSV), closely related to rabies virus, has served as a paradigm to study the fundamental molecular mechanisms of transcription and replication of NNS RNA viruses. These studies provided insights into how NNS RNA viruses synthesize 5′-capped mRNAs using their RNA-dependent RNA polymerase L proteins equipped with an unconventional mRNA capping enzyme, namely GDP polyribonucleotidyltransferase (PRNTase), domain. PRNTase or PRNTase-like domains are evolutionally conserved among L proteins of all known NNS RNA viruses and their related viruses belonging to Jingchuvirales, a newly established order, in the class Monjiviricetes, suggesting that they may have evolved from a common ancestor that acquired the unique capping system to replicate in a primitive eukaryotic host. This article reviews what has been learned from biochemical and structural studies on the VSV RNA biosynthesis machinery, and then focuses on recent advances in our understanding of regulatory and catalytic roles of the PRNTase domain in RNA synthesis and capping.Non-segmented negative strand (NNS) RNA viruses belonging to the order  Mononegavirales comprises highly diversified eukaryotic viruses with a monopartite negative strand RNA genome (rarely bipartite genomes), which includes important human pathogens  , and Filoviridae , Bornaviridae, Nyamiviridae, Mymonaviridae, Sunviridae, Artoviridae, Lispiviridae, and Xinmoviridae (The order  (EBOV)] . Since goviridae . Over thoviridae .Vesiculovirus genus in the Rhabdoviridae family. VSV has served as a prototype to elucidate the fundamental molecular mechanisms of transcription and replication of NNS RNA viruses, since its virion-associated RNA-dependent RNA polymerase (RdRp) activity was discovered ] is an arthropod-borne animal virus belonging to the scovered . A bullescovered . An RdRpscovered , and is scovered . In the scovered , which iscovered .N, P, M, G, and L, which are arranged in tandem from the 3′- to 5′-end , Paramyxoviridae , Pneumoviridae , Filoviridae , Bornaviridae , Nyamiviridae , and other families share the same gene organization with VSV, but have diversified their structural genes and often acquired additional structural and/or non-structural genes during evolution , Newcastle disease virus (Paramyxoviridae), MeV, HRSV], VSV has proven to be a remarkable model to elucidate the mechanisms of RNA biogenesis by NNS RNA viruses. Early studies using in vitro transcription systems with detergent disrupted VSV particles and purified RNPs revealed that VSV packages all enzymes required for primary transcription into virions, including RdRp  proteins are catalytic and regulatory subunits, respectively, of the RdRp complex  and 5′-trailer (Tr) sequences, respectively, and contains the five internal genes that are tandemly connected via intergenic regions  of ∼47 nt ppp(5′)Am-: N7-methylguanosine(5′)triphospho(5′)2′-O-methyladenosine]  and P proteins  accumulated in infected cells play a key role in co-replicational encapsidation of the antigenome or genome  is a capsid protomer for the intact nucleocapsid, the assembled N/RNA polymer . StudiesMedium resolution reconstructions from electron microscopy studies of the VSV  and RABVThe VSV and RABV N protein structures also revealed detail on how RNA is stored by the capsid as well as the unique structure adopted by the RNA upon encapsidation. When the N protein subunits assemble to form the nucleocapsid, they generate a continuous tunnel that sequesters the RNA upon encapsidation and protecting the RNA against degradation by nuclease and base-catalyzed hydrolysis . The N- 0–P complex, the N-terminal helical region of P (residues 17–31) interacts with the central hinge region of N  (The VSV P protein (265 amino acids) is a dimeric, non-globular molecule containing structured domains and disordered regions . During  complex . The N-tes 6–35)  and the es 6–35) , while ies 6–35) . The dimes 6–35) .0   of recombinant VSV L protein was reported to initiate transcription from a naked RNA template with a 19-nt VSV Le promoter sequence (0.2 μM) in the absence of the P protein   resides ongation . In addiongation  is requinthesis) . Althougnthesis) . Thus, tnthesis)  appears  protein , althoug protein .Non-segmented negative strand RNA viral L proteins share six conserved regions  , in whicThe first electron microscopic (EM) analysis of the VSV L protein  revealed that it consists of an N-terminal ring-like structure and a flexible C-terminal appendage containing three globular domains, which were predicted to be responsible for RNA synthesis and cap formation, respectively . The P pC)   of the VSV L protein at 1.8 Å resolution showed that residues 43–371 constitute a flat domain  composed of two subdomains (I and II) . The NTDC) , the PACV RdRp L , and theV RdRp L , suggestV RdRp L . Some coV RdRp L . In our a-5k432) , D290 ani). Although there is a possibility that amino acid residues in the RdRp domain serve as a general base  and a general acid  to mediate deprotonation of the 3′-OH group and protonation of leaving PPi, respectively, as proposed for other polymerases -dependent MTase core fold with a glycine-rich SAM binding motif G[-]GxG  and a 2′-O-MTase motif, namely K–D–K–E catalytic tetrad  (N7 and nucleoside1 (N1)-2′-O positions (1-2′-O-MTase (VP39), the second lysine residue in the K–D–K–E tetrad may play a critical role in cap-specific N1-2′-O-methylation by steering the 2′-OH oxygen orbital toward the methyl group of SAM  . Similar5–E1833) , the sinositions . As suggp of SAM  rather tp of SAM .N7-methylguanosine (m7G) linked to the first nucleoside (N1) of mRNA through an inverted 5′-5′ triphosphate bridge (ppp). Lower eukaryotic mRNAs have an m7GpppN1- cap structure , whereas the cap structure of higher eukaryotic mRNAs is further methylated at 2′-O positions to various degrees into m7GpppN1m- (cap 1) and m7GpppN1mpN2m- (cap 2)  is sequentially formed on pre-mRNA by mRNA capping enzyme with the RNA 5′-triphosphatase (RTPase) and mRNA guanylyltransferase (GTase) activities followed by a cap-specific MTase(s) (reviewed in i). (2) GTase transfers a GMP moiety of GTP (GMP donor) to ppRNA (GMP acceptor) via a covalent enzyme-(lysyl-Nζ)–GMP intermediate to form GpppN1-RNA with concomitant release of PPi. (3) mRNA (guanine-N7-)-methyltransferase (G-N7-MTase) transfers a methyl group from SAM to GpppN1-RNA to generate m7GpppN1-RNA (cap 0-RNA) and a byproduct, S-adenosyl-L-homocysteine (SAH). (4) In higher eukaryotic cells, mRNA (nucleoside1-2′-O-)-methyltransferase (N1-2′-O-MTase) methylates cap 0-RNA to yield m7GpppN1m-RNA (cap 1-RNA) (2-2′-O-)-methyltransferase (N2-2′-O-MTase) methylates cap 1-RNA to produce m7GpppN1mpN2m-RNA (cap 2-RNA) -methyltransferase (Am1-N6-MTase) often methylates the cap 1 structure to generate m7Gpppm6Am-RNA   . (5) Furp 2-RNA) . (6) If enosine) . m6A metof mRNAs , althougof mRNAs .N7-MTase methylates GTP to produce m7GTP. (3) mRNA N7-methylguanylyltransferase (m7GTase) transfers m7GMP from m7GTP to ppRNA via a covalent enzyme–m7GMP intermediate to generate m7GpppN1-RNA (cap 0-RNA). Interestingly, although alphaviruses do not have N1-2′-O-MTase, a structural element within the 5′-untranslated region of their cap 0-RNA genome confers resistance to the IFIT1-mediated restriction (Saccharomyces cerevisiae viruses L-A and L-BC) have a new class of mRNA m7GTases. Totiviral m7GTase transfers an m7GMP moiety of host m7GpppRNA to viral ppRNA through a covalent enzyme-(histidyl-N)-m7GMP intermediate to generate viral m7GpppN1-RNA, thereby causing decapping of host mRNAs  of mRNAs (1 (A1)-2′-O position followed by the G-N7 position to sequentially generate GpppAm and m7GpppAm (cap 1)  . Therefo7GMP) transfer mechanisms employed by eukaryotes and other viruses (Vesiculovirus) (Lyssavirus) (Nε2)–pRNA (L–pRNA) intermediate to yield GpppA-RNA in an mRNA-start sequence dependent manner (Km for GDP (0.03 μM)  , and RABsavirus) , use a psavirus) . In the savirus) , 2008. Gt manner . PRNTaset manner . Howevert manner  that are0.03 μM) . In addie pppRNA , althoug1R2C3) and the fifth residue (G5) are essential and important, respectively, for the pRNA donor substrate activity  and higher (10 μM) Km values for SAM, respectively, co-transcriptionally methylate the cap structure on mRNAs, producing GpppAm in the presence of limited concentrations (< 0.1 μM) of SAM and m7GpppAm  in the presence of higher concentrations (> 5 μM) of SAM  and minor (GpppA- → m7GpppA- → m7GpppAm-) pathways  under the reported conditions, although it was fully capable to generate m7GpppAm on mRNAs co-transcriptionally when using our reconstituted transcription system (Using the VSV ) of SAM . In addim7GpppAm . It is ipathways . Since tpathways . Rahmeh n system . The reain vitro transcription reactions is known to induce production of mRNAs with an extremely long poly(A) tail  tail . Similar(A) tail  as well (A) tail  of the V(A) tail . These oParamyxoviridae) L protein is enough to specifically methylate a capped 5-nt RNA with the SeV mRNA-start sequence (GpppAGGGU) at the G-N7 position, but not at the A1-2′-O position, to produce ∼10 fmol of m7GpppA within 2 h -2′-O-methylation similar to eukaryotic cap methylation. In contrast, recombinant C-terminal fragments of the L proteins of human metapneumovirus (HMPV) (Filoviridae)  of a recombinant form of the SeV  , closelyviridae)  were repin vitro , althoug7Gppp(m6)A1mp(m6)A2(m)-, where A1 is predominantly m6Am and A2 is A, Am, or m6Am  are present in the VSV PRNTase domain Wx(3–8)ΦxGxζx(P/A) , (Y/W)ΦGSxT (motif B), W (motif C), HR (motif D), and ζxxΦx(F/Y)QxxΦ (motif E) . These mfamilies . Motifs families , forminge domain , but aree domain . The dome domain , which ie domain .Nε2 position of H1227 in the VSV PRNTase domain via a phosphoamide bond  of the VSV L protein were originally identified as essential for the formation of a product sensitive to tobacco acid pyrophosphatase, most probably 5′-terminal GpppA cap, on RNA by alanine scanning mutagenesis , althougide bond . H1227 iide bond . A propoide bond  requiresOur extensive mutagenesis analysis further identified G1100 in motif A, T1157 in motif B, W1188 in motif C, and F1269 and Q1270 in motif E as essential or important for the PRNTase activity in the step of the intermediate formation . These kOγ1 atom of T1157, while phosphate three is positioned to interact with the side chain of Q1270. The adenine base punches into a second cavity on the opposite side of the cleft from the guanine. F1269 sits to one side of the entrance to this cavity and is in position where by rotation of the side chain could π-stack with the adenine. The adenine base is also positioned ∼3 angstroms from the side chains of N1264 and S1224, which could interact with atoms N6 and N7, respectively. The active site histidine, H1227, sits between phosphate two and three of the cap analog.To speculate functions of the VSV PRNTase domain in the cap formation, we performed a docking study with a GpppA cap analog corresponding to a 5′-terminal part of a capped RNA product (GpppAACAG-) and found that it fits in a crevice adjacent to motifs B–E . The guain vitro RNA capping as well as replication of recombinant VSV in host cells  in the intermediate formation step, and can be replaced with histidine yielding partial activity, but not with lysine, in the pRNA transfer reaction , D (H1227 and R1228), and E  . The arost cells . Howeverormation   extended from the PRNTase domain into the RNA exit cavity of the RdRp domain . The pri capping . In conte domain .De novo initiating RdRps of other unrelated viruses  often have a “priming loop,” which facilitates primer-independent transcription initiation with initiator and incoming nucleotides by stabilizing their initiation complex formed at the 3′-terminal of their genomic RNAs  at the 3′-terminal UG sequence of the e genome . Based oe genome  and the e genome , the VSVe genome . In thisage RdRp , the tryde novo initiation  of the tryptophan residue (W1167) on the VSV priming-capping loop is not required for internal initiation  position, releasing a 5′-triphosphorylated abortive N mRNA fragment   in the gene-start sequence, but prematurely terminates transcription, resulting in production of uncapped transcripts of 40–200 nt . Since 5d N1–40) . TherefoN gene, producing unusual N mRNA fragments initiated with GTP , such as an internal fragment with residues 41–68 (N41–68) and 3′-polyadenylated fragment with residues 157–1326 (N157–1326)  transcripts of heterogeneous lengths (100 to 500 nt). Furthermore, these mutations were reported to repress 3′-polyadenylation of transcripts when using their reconstituted transcription system in the presence of rabbit reticulocyte lysates , putative PRNTase inhibitors as well as mutations in a putative PRNTase domain of the L protein also cause premature termination of mRNA synthesis, resulting in production of 5′-uncapped abortive transcripts (< 50 nt)  . It shoutive VSV  had alream mRNAs . In contam mRNAs , 2009 sh lysates , 2009. H lysates , the cap< 50 nt) . Thus, iRhabdoviridae, Paramyxoviridae, Pneumoviridae , Filoviridae, Bornaviridae, and Nyamiviridae families possess a PRNTase or PRNTase-like domain with the conserved motifs A–E  belonging to the Chuviridae family in the order Jingchuvirales  (see Mymonaviridae), and 6 miviruses , while motif E is absent in those of all known miviruses. Interestingly, L proteins of 4 novirahbdoviruses  see . As in town) see , motif Coviruses  and 22 moviruses , therebyNucleorhabdovirus genus of Rhabdoviridae, Bornaviridae, and Nyamiviridae have a PRNTase-like domain, but lack an MTase domain  in PRNTase and PRNTase-like domains of selected viruses . The phye domain , we suggAs described in this review article, the numerous biochemical and structural studies on the VSV RNA biosynthesis machinery have led to the remarkable discoveries, provoking paradigm shifts in understanding unique roles of NNS RNA viral proteins in transcription and replication. Those include the finding that the rhabdoviral L proteins catalyze the unique mRNA capping reaction by the unconventional mechanism involving their GTPase and PRNTase activities. The striking differences between host and viral mRNA capping systems emphasize the potential of NNS RNA viral PRNTase domains as attractive targets for developing anti-viral agents.in vitro transcription or capping systems for other NNS RNA viruses has hampered the progress in our understanding of their precise mechanisms of mRNA capping. Our hypothesis is challenged by studies on some paramyxoviral L proteins that suggest that their C-terminal portions may carry out conventional mRNA capping with their RTPase and GTase activities . These studies would provide definitive evidence to distinguish between unconventional and conventional capping mechanisms. Unfortunately, this approach cannot be applied to 5′-G-started mRNAs of NNS RNA viruses, such as HRSV  enzymes  and/or other impurities that affect the reactions, and their enzymatic products are unambiguously identified using appropriate methods. It is also very important to demonstrate that these recombinant proteins exhibit enzymatic activities and substrate specificities that are the same as or similar to those of their native forms, if available. The classical approach to perform e of VSV , it woul as HRSV . NeedlesIt is now apparent that rhabdoviral PRNTase domains perform covalent catalysis in the pRNA transfer reaction to GDP to produce the cap structure . Howeverde novo initiation by L proteins of other NNS RNA viruses and identify their priming elements, if any. Detailed investigation along these lines would certainly advance our understanding on how mononegaviral RNA biosynthesis machineries play common and virus-specific roles in transcription and replication at the molecular level, and eventually reveal an Achilles’ heel for a target in developing anti-viral agents.Non-segmented negative strand viral L proteins may have retained evolutionary conserved elements critical for common functions, but diversified some other regions for virus-specific functions. Thus, the important studies would be to explore virus-specific functions of respective NNS RNA viral L proteins. Since mRNAs of NNS RNA viruses belonging to different families are unique due to virus-specific mRNA-start sequences that are distinct from those of rhabdoviruses , it woulTO conceived and wrote the manuscript, and prepared the figures. TG wrote the part of the manuscript, performed the docking study, and prepared the structural images.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nature, Chen et al.1 identified the critical transcription factors that drive T-cell dysfunction. Knockout of these transcription factors to augment T-cell activity represents a new approach to enhancing the effectiveness of chimeric antigen receptor T (CAR-T) cell therapy for solid tumors.In a recent report in 2 Therefore, intensive efforts have been made to augment the efficiency of CAR-T cell therapy for solid tumors.CAR-T cells are designer cytotoxic lymphocytes with recognition specificity and tumoricidal potency. Although CAR-T cells exhibit unprecedented effectiveness in treating hematologic malignancies, these cells have limited efficacy against solid tumors.3 Recently, CAR-T cells armed with a secreted minor fragment derived from the variable domains of a PD-1-blocking antibody were tested.4 This design seemed more efficient to augment the functionality of host and nearby T cells than did the full-length antagonist. Soluble transforming growth factor-β (TGF-β) is also important for inhibiting T-cell activation. By introducing signal-switch receptors comprising the TGF-β-binding ectodomain and the T-cell activation endodomain, CAR-T cells are more potently activated in the tumor microenvironment.5 The suppressive immune cells represent another obstacle for CAR-T cell therapy. With enforced expression of interleukin (IL)-18, CAR-T cells are more active against tumors by decreasing the populations of suppressive regulatory T (Treg) cells and M2 macrophages.6A major barrier in solid tumors is the hostile microenvironment surrounding CAR-T cells. To overcome the immunosuppressive effects within the tumor niche, several tactics have been undertaken. The administration of programmed cell death protein 1 (PD-1) antibodies is effective in restoring CAR-T cell function.7 Similarly, supplementation with a phosphoinositide 3-kinase-δ (PI3k-δ) antagonist shows promise in preserving memory subsets during CAR-T cell expansion.8 Alternatively, an infusion with naive T cells, which subsequently differentiate in vivo, may result in better clinical responses. The combination of CAR-modified induced pluripotent stem cells (iPSCs) and organoid-dictated T-cell lineage commitment makes this hypothesis possible. A previous group established such an approach to produce naive CAR-T cells,9 which may introduce new scenarios in treating tumors.T-cell differentiation is also the determinant for therapeutic efficacy. Less differentiated T cells have better persistence in vivo. Nevertheless, a large portion of T cells are terminally differentiated during CAR-T cell production. To augment the populations of more proliferative T cells with stem-cell-like or memory phenotypes, specific supplements were added in culture. The addition of IL-7 and IL-15 but not IL-2 induces the generation of more memory cells without compromising the cytotoxic function.Nature proposed a new strategy to improve CAR-T cell therapy.1 To avoid autoimmunity, various transcription factors are upregulated to limit the intensity and interval of T-cell activation. Such factors will upregulate the expression of inhibitory receptors, subsequently induce terminal differentiation and downregulate the secretion of inflammatory cytokines. Hence, finding the key intrinsic initiator of T-cell dysfunction can provide new targets for the preparation of upgraded CAR-T cells. Chen et al.1 uncovered NR4A1, NR4A2 and NR4A3 as the central transcriptional factors that drive T-cell dysfunction. To identify the intrinsic triggers of exhaustion, these authors checked the transcriptional adaptations between functional and hypofunctional CD8+ T cells. By analyzing single cell transcriptome and chromatin accessibility, the researchers discovered that the transcription factors NR4A1, NR4A2 and NR4A3 were upregulated and played critical roles in mediating CD8+ T-cell exhaustion in mouse CAR-modified and endogenous lymphocytes and their human counterparts exposed to tumor or chronic virus infection. When all three NR4As were deficient, the activated CD8+ T cells demonstrated features resembling effector T cells with increased functionality rather than those with an exhausted status. Additionally, the researchers generated CAR-T cells with a deletion of NR4As. Compared with their wild-type counterparts, the NR4A-deficient CAR-T cells showed enhanced cytotoxicity but decreased inhibitory receptor expression and promoted tumor regression more significantly.Recently, a report published in This finding provides us with new candidate targets to augment the cytotoxic capacities of CAR-T cells during chronic antigen engagement, which supplements the approaches that help to improve the effectiveness of adoptive transfer therapy for solid tumors Fig. .Fig. 1St


Drosophila, and vertebrate models such as mice and rats. In each case, germ stem cells undergo mitotic division to proliferate and then continue, via meiosis, through various stages of elongation and individualization from spermatogonia to spermatid to finally to form mature sperm. Mature sperm are then stored in the seminal vesicles for fertilization. Errors in any of these stages can lead to male infertility. Here, we identify that Drosophila Pif1A acts as a key regulator for sperm individualization. Loss of Pif1A leads to male sterility associated with irregular individualization complex and empty seminal vesicles without mature sperm. Pif1A is highly expressed in the testes of mated male adult flies and the Pif1A protein is expressed at a higher level in male than in female flies. Pif1A is homologous to mammalian coiled-coil domain-containing protein 157 (CCDC157), which is also enriched in the testes of humans and mice. Human CCDC157, with unknown function, was identified to be downregulated in men with idiopathic non-obstructive azoospermia (NOA). We map the function of Drosophila Pif1A during spermatogenesis, showing that Pif1A is essential for spermatide individualization and involved in the regulation of the lipid metabolism genes. Our findings might be applicable for studying the function of CCDC157 in spermatogenesis and other aspects of human male fertility.The dynamic process of spermatogenesis shows little variation between invertebrate models such as  Drosophila in insects to mice and rats in mammals1. Each developmental stage is precisely controlled by intrinsic signals. Human male infertility results from abnormal spermatogenesis and is mostly due to chromosomal alterations, Y chromosome microdeletions, and related gene mutations2. For many of these, the detailed mechanisms are often technically inaccessible and remain obscure. In Drosophila, the testes are easy to dissect and the morphological changes during each stage of spermatogenesis can be easily visualized3. Drosophila is, therefore, well established as an excellent model for the study of spermatogenesis4. Recently, a large-scale RNAi screen in Drosophila testes has been conducted to analyze the genes required for germline stem cell (GSC) maintenance or differentiation5. Many of these genes were noted as those involved in key steps of protein synthesis and degradation relating to GSC homeostasis7, while others were identified as key regulators of the dynamic process of sperm morphogenesis9. However, the molecular links between different stages of spermatogenesis remains largely unidentified. The combination of various genetic techniques applied to Drosophila, such as CRISPR/Cas910 and the Gal4/ UAS transgenic system11, have enabled the convenient mapping and prediction of functional genes which facilitate, for example, the identification of genes that might cause infertility due to deficiencies in a specific developmental stages or in certain type of cells.Spermatogenesis is a highly conserved process, not differing appreciably between any animal model, from Drosophila testis. A gene of unknown function, PFTAIRE interacting factor 1A (Pif1A)12, was of particular interest. The female mutants of Pif1A displayed normal fertility but the males were infertile. Pif1A is the only homolog of the mammalian coiled-coil domain-containing protein 157 (CCDC157) family (www.pantherdb.org). Pif1A has been noted to exist at a high transcriptional expression level in the testes of mated male flies (www.flybase.org) and the Pif1A protein is expressed at a higher level in male than in female flies12. Notably, CCDC157 has also been observed as highly expressed in the testes of humans and mice, and to be downregulated in men with idiopathic non-obstructive azoospermia (NOA)14. Here, we map the function of Pif1A during Drosophila spermatogenesis, which might be informative for human CCDC157.To gain insight into the molecular basis for male sterility, we made use of CRISPR/Cas9 to produce mutations of the genes shown to be highly expressed in Drosophila adults contain a pair of testes, each testis is a coiled tube with a closed apical end and a basal end that connects to the seminal vesicle. At the apical end there are approximately 8–12 GSCs. Spermatogenesis takes place within individual units known as cysts15, where GSCs divide asymmetrically in the cysts to give rise to spermatogonial cells. Each GSC is flanked by two somatic cyst stem cells (CySCs) that eventually differentiate into a head cyst cell and a tail cyst cell, analogous to mammalian Sertoli cells17. Spermatogonia then go through four mitotic divisions, generating 16 primary spermatocytes that further undergo two meiotic divisions to yield a group of 64 syncytial haploid spermatids. Round spermatids are inter-connected by abundant cytoplasmic bridges18. During post-meiotic spermatid differentiation, syncytial cysts of 64 haploid spermatids undergo synchronous differentiation. Numerous changes occur at a subcellular level including formation of flagellar axonemes and acrosomes, remodeling of mitochondria and nuclei, and the polarization of elongating cysts and the plasma membrane6. The fully elongated syncytium of 64 spermatids undergoes a membrane remodeling process known as individualization19.Male 18. These investment cones assemble into a macroscopic structure referred to as the individualization complex (IC), from which the cyst membrane is remodeled and intercellular bridges are resolved to encase each sperm cell in its own plasma membrane19. The IC moves processively from the heads to the tips of the tails along the spermatid bundle20. During this process unneeded organelles, mitochondrial DNA, and cytoplasmic components are stripped away, forming the observable dilation of the cyst known as the “cystic bulge”. The cystic bulge is then detached at the tip of the tail where it becomes known as the “waste bag”21.Individualization begins with the formation of investment cones around each of the 64 needle-shaped nuclei23. In myosin V mutants, fewer actin cones were able to form24. Myosin VI acts to stabilize the actin cones and the Arp2/3 complex is required for the formation of the actin meshwork27. Cortactin co-localizes with Arp2/3 complex and myosin VI at the leading edge of the actin cone during IC movement28.Individualization results in the formation of individual sperm. The structure of the actin cones and their cohort movement is critical for spermatid individualization. Cytoskeletal regulators, such as myosin V, myosin VI, cortactin, and Arp2/3 complex, have been identified to influence the formation of actin cones and the synchronous movement of the IC21. Multiple Drosophila caspases and caspase regulators participate in this process. Mutation of any of these genes leads to individualization defects9. During complex membrane remodeling, lipids including phospholipids, sphingolipid and cholesterol, act as essential membrane structural components and also play critical roles. Lipid regulators including Drosophila nessy (nes), oysgedart (oys), noa, oxysterol binding protein (osbp), fan, npc1 have been identified32, although the role of most of their protein products remains obscure. The mutation of osbp, for example, leads to abnormal sterols trafficking and distribution and in corresponding male infertility30. The regulation mechanism of OSBP remains unclear.The individualization process has also been defined as a caspase-dependent apoptosis-like event, requiring functional proteasomes and membrane traffickingPif1A is essential for spermatid individualization and might act to regulate lipid metabolism genes during spermatogenesis. Loss of Pif1A results in the downregulation of several lipid regulators and in a phenotype that genetically mirrors that of the loss of osbp. By dissecting the testes and conducting immunofluorescence staining, we found that Pif1A mutants showed normal GSCs divisions and spermatid elongation but displayed disorganized IC and irregular IC movement. Correspondingly, no mature sperm could be observed in their seminal vesicles. The duplication of the Pif1A genome could partially rescue this phenotype. We show that Pif1A plays a critical role during spermatogenesis and thus is essential for male fertility. Our findings might be applicable for studying the function of CCDC157 in spermatogenesis and in other aspects of human male fertility.In this study, we have identified that www.flybase.org). We focused on Pif1A and identified that one of the mutations, Pif1A1, resulted in compromised male fertility but left female fertility unaffected. Expression of Pif1A mRNA in WT adult testis is clarified by whole-mount in situ hybridization  by quantitative PCR (qPCR). The mRNA levels were significantly reduced in Pif1A1 and Pif1A1/DF male animals, indicating Pif1A1 is a loss-of-function mutation . The duplication of the Pif1A genome was able to partially restore the male reproductive capacity  and waste bags (WBs) could be visualized with antibodies specific for activated DrICE . In the WT, very little cytoplasm was left in individualized sperm and that each axoneme/mitochondria pair was tightly surrounded by a clear integral plasma membrane Fig.  and the asm Fig. , althougmal Fig. . The neb WT Fig. . The mRNted Fig. . These dDrosophila, the mammalian homolog of the protamines and transition proteins were identified as ProtamineA/Protamine B and Tpl94D50. We constructed ProtB-eGFP in Pif1A1 mutants background and evaluated the GFP patterns. ProtB-eGFP did not express in young elongating nuclei and early canoe stage spermatids but begun expression from the late canoe stage spermatids onwards50. The GFP signals in late canoe stage spermatids were detected in both Pif1A1 and WT animals . We confirmed that loss of Pif1A neither effect on germ cell division nor spermatid elongation, but sperm maturation.In this study, we have applied Drosophila, the Pif1A gene has five annotated transcripts, however, only the Pif1A-RG and Pif1A-RI are highly expressed in the testes of WT animals with Pif1A-RH and Pif1A-RJ being barely expressed  could partially restore the male reproductive capacity BSC478/TM6(BL24982), Dj-GFP (BL5417), Pif1A-GFP.FPTB/Cyo(BL42670), dj-GFP(BL5417), protamine B-eGFP/Cyo(BL58406); bam-Gal4 and tj-Gal4 (kindly provided by Chao Tong); nos-Gal4 and UAS-actin.GFP/CyO; Sb/TM6B (Tsinghua Fly Center).Flies were reared on standard cornmeal medium at 25 ℃. The following strains were used: wPif1A mutants, we employed the “CRISPR/Cas9 method”, the gRNA was designed to recognize a 19-nt target sequence and act to direct Cas9-mediated cleavage of both DNA strands within the target site.To generate the Pif1A cDNA with the primers below and cloned it into the pUAST-attb vector. These constructs were then transformed into VK33 embryos using the standard P-element-mediated transgenesis protocol.To generate UAS-Pif1A-RI, UAS-Pif1A-RG, and UAS-eGFP-Pif1A-RG transgenenic flies, we amplified the full length Pif1A- RG-FCTGCGGCCGCGGCTCGAGATGGCTGAAAACCAAACCAAAACGPif1A- RG-RTCACAAAGATCCTCTAGATCAGAGCCGGGCATTCTCGGACGGPif1A- RI-FCTGCGGCCGCGGCTCGAGATGGGCAACGAGGAATCCTPif1A- RI-RTCACAAAGATCCTCTAGACTATTTCTTAGCTCTGAACAAGeGFP-Pif1A- RG-F TTCGTTAACAGATCTGCATGGTGAGCAAGGGCGAGGAeGFP-Pif1A- RG-R ATCTCGAGCCGCGGCCGCCACTTGTACAGCTCGTCCATGOne-day-old males (1 day) from different genotypes were arranged to mate with wild-type 3–5-day-old virgin females in small cages  for one day before calculating the embryo hatch rate. Embryos were then collected on new apple juice agar plates for 4 h and incubated at 25 °C and 45–70% humidity for 24 h. Hatch rates were determined from the proportion of hatched eggs to total eggs (200 each time randomly selected from the plates). Under microscopy, one can easily distinguish the hatched embryos showing shriveled egg shells from the unhatched eggs with the appearance of plump grains of white rice. This experiment was repeated three times.g for 10 min at 4 ℃. Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene fluoride membrane. Membranes were immunoblotted with the primary antibodies and then probed with the secondary antibodies. Rabbit anti-cleaved Caspase 3  and mouse anti-tubulin  were used as the primary antibodies. Blots were treated with the ChemiLucent ECL detection reagents (Millipore) and protein bands were visualized using the Chemiluminescence Imaging System .Protein extracts from testes were prepared by grinding tissue in lysis buffer  containing the protease inhibitor cocktail (Roche). The lysates were cleared by centrifugation at 14,000 x Five days after eclosion the testes were dissected from adult males and prepared for electron microscopy using standard protocols. Thin sections were observed and photographed using a Hitachi H-7650 transmission electron microscope.® First-Strand cDNA synthesis kit. We used Power SYBR Green PCR Master Mix (Applied Biosystems) to conduct RT–PCR with rp49 used as control. Real-time PCR was performed on an ABI7900HT Fast real-time PCR machine. The PCR primers used are provided in below;Animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Zhejiang University. Total RNA from was extracted using RIzol reagent (Invitrogen) from 5-day-old adult testes. The first-strand cDNA was synthesized from 2 μg of total RNA samples using an Invitrogenrp49-qf GCTAAGCTGTCGCACAAA, rp49-qr TCCGGTGGGCAGCATGTG;Pif1A-all-qf CAATGCCAGCTCTTGGAACG, Pif1A-all-qr AATCACGACCCTCGAGATC;pif-RA-qF AAATCAAATGGTGAAATTAC,pif-RA-qR GTTGGGTTATCTTGACATCC;pif-RI-qF TCTGGACTGTGTCCATAACC, pif-RI-qR ACAAGGTGGAATTGCTCCTC;pif-RJ-qFAATAATTTCCCCTGTGTTGA, pif-RJ-qR GATTCTCCTCAATAAATTTG;pif-RH-qF GTTGCCCAAGACTCCTCTTA, pif-RH-qR TCTCCTCAATTATTTGCATT;pif-RG-qF CTGTTTACATTACAACATCG, pif-RG-qR TCTCCTCAATCATTAGCTCT;ark-qf GCCTGCAACCGTATCTGAAGCTAA, ark-qr TCTTCAAGTTAAGCCAGAAGATC; dronc-qf GCGTTAGCAAGCTCCGGAATGA, dronc-qr CCTCGGTTGAAACGTGATTGC;Nessy-qfATTCTGGCAGGTTATCCGGT, Nessy-qrGGACGTGTAGAAATATCCCA;Oys-qfTTGTCCAAAGAGCCGTCCTG, Oys-qrCTAGGATGCTTTGGAAGTGC;Noa-qfTCGGCCTAGATTCCAATTGC, Noa-qrGGTGATGTGGTGATACCAGT;Osbp-qfGTACGTTCAAATCAATCCCT, Osbp-qrCCACTTGACCTCGTTGTCTT;Fan-qfCTTTGAGGGACCATTCAACC, Fan-qrCTGCATCTTCCAAAACTCCT;Ccdc157-qf GGAGGAGAACAAGCGGCTCC, Ccdc157-qr GCCTGTCAACCCTGAGAATG;L17-qf CGGTATAATGGTGGAGTTG; L17-qr ACCCTTAAGTTCAGCGTTACT.t-test. For the white light images of testes, 5-day adult males were dissected in PBS and placed on a glass slide containing a drop of PBS at 25 °C then obtained using a Nikon SMZ1500 microscope.The testes of 5-day adult males were dissected in phosphate-buffered saline (PBS). They were then fixed in PBS containing 4% formaldehyde for 15 min at room temperature and washed three times for 15 min each in PBS containing 1% Triton X-100 (PBT) before being blocked for 1 h in PBT containing 5% BSA. The samples were then incubated with primary antibodies  overnight at 4 ℃ followed by four 15 min washes in PBT. The samples were incubated with secondary antibodies Alexa Fluor 555 -conjugated  for 1 h at room temperature. Phalloidin staining was performed using Alexa Fluor 488 (Invitrogen) (1:500) for 30 min at room temperature. 4’, 6-diamidino-2-phenylindole (DAPI)  was added to stain the nuclei. Tissues were mounted and viewed. The slides were observed and photographed using an Olympus FV1000 confocal laser scanning microscope and processed using Adobe Photoshop. To quantify the number of ICs in each testis, a confocal Z series of the testes were acquired at a step size of 1.5 mm. For the measure of nebenkern size, images were processed with ImageJ  to measure the relative areas of nebenkern as an indicator of the size and data were analyzed with a Student’s Pif1A1 mutants were dissected in Schneider’s medium and observed under a Nikon Eclipse 80i fluorescence microscope with a 20 objective lens. Images were acquired at 29.97 frames per second.For live imaging, the testes of 3 day-old adult males from control animals and https://www.ebi.ac.uk/Tools/msa/clustalo/. Protein structure prediction analysis was performed using SWISS-MODEL (https://swissmodel.expasy.org/) and visualized by UCSF Chimera program. We calculated and generated the plots underlying the differences of CCDC157 mRNA expression levels in nine types of mouse and human tissues respectively, and in 16 patients with NOA compared with four men with normal spermatogenesis by performing the one-way Analysis of variance (ANOVA) by GraphPad Prism 6 . The expression levels of CCDC157 were analyzed by Mann–Whitney test.Alignment analysis was performed using 64. Digoxygenin (dig)-labeled RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; Pif1A probe was generated by using Pif1A-RG mentioned above as the template with primers Pif1A-F, GGCTACACTTCTTCTATACA; Pif1A-R TACGACTCACTATAGGATTTTGTTCTCCAGCGTG. The antisense probe was generated by using primers Control-F TAATACGACTCACTATAGGCTACACTTCTTCTATACA; Control-R GGATTTTGTTCTCCAGCGTG. The Pif1A probe was the five annotated transcripts common sequence. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction.Whole-mount adult testis RNA in situ hybridization (ISH) was performed as describedSupplementary informationMovie 1Movie 2 ICs-Pif1AMovie 3 CB-WTMovie 4 CB-PIF1A


Salicylidene thiosemicarbazones, of general formula (X)N-NH-C(S)-NH(Y), were prepared through the condensation reaction of 2-hydroxybenzaldehyde and its derivatives (X) with thiosemicarbazide or 4-phenylthiosemicarbazide . The characterization of the new formed compounds was done by 1H-NMR, 13C-NMR, IR spectroscopy, elemental analysis, magnetochemical, thermoanalytical and molar conductance measurements. In addition, the structure of the complex 5 has been determined by X-ray diffraction method. All ligands and metal complexes were tested as inhibitors of human leukemia (HL-60) cells growth and antibacterial and antifungal activities.Thirty two new Cu(II), Ni(II) and Zn(II) complexes ( In-situ one-pot template condensation reactions lie at the heart of the coordination chemistry. Transition metal complexes have also received great attention because of their biological interests, including antiviral, anticarcinogenic, antibacterial and antifungal activities  using successive dilutions method ,37,38. Sreported .in vitro on laboratory stems: Aspergillus niger, Aspergillus fumigatusi, Candida albicans and Penicillium. The activity has been determined in liquid Sabouroud nutritive environment (pH 6.8). The inoculates were prepared from fungi stems which were harvested during 3–7 days. Their concentration in suspension is (2–4) × 106 colonies form unities in milliliter. Sowings for levures and micelles were incubated at 37 °C during 7 and 14 days, respectively.Antimycotic properties of the complexes were investigated 5 has been determined by single crystal X-ray diffraction method. The IR, 1H-NMR and 13C-NMR data were successfully used to elucidate the formation of the salicyliden thiosemicarbazones ligands. All ligands and their metal complexes were tested as inhibitors of HL-60 cells proliferation. The ligands have unsignificant inhibitor activity at 0.1 and 1.0 μmol/L, but at 10 μmol/L 2L8H, 2L9H and 2L10H inhibit the cell proliferation. The copper complexes, including inner sphere water and tridentate ONS ligands, are more active than those containing inner sphere amine, which blocked the metal active centre. The most indicative criteria for future synthesis of biological active coordination compounds from theview point of the inhibition of HL-60 cell proliferation, antibacterial and antifungal activity: use of copper (II) complexes and presence of sulphur atom in the tridentate organic ligand.Ten new salicyliden thiosemicarbazones ligands and their corresponding Cu(II), Ni(II) and Zn(II) complexes have been synthesized and characterized. The molecular structure of the complex


Objective: Macrophage Migration Inhibitory Factor (MIF) is involved in the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). MicroRNAs (miRNAs) play important roles in LN but whether specific miRNAs regulate the expression of MIF in LN is unknown. We explore specific miRNAs that can regulate MIF expression, and investigate miR-654 for the treatment of experimentally-induced murine lupus nephritis.Methods: Sera samples from 24 SLE patients and 24 controls were collected to measure the MIF concentration and its correlation with disease activity. A luciferase reporter assay was used to explore the target of miR-654. ELISA was used to detect the downstream cytokines regulated by miR-654 and MIF. Western blot was applied to measure the impact of miR-654 inhibition on downstream MIF signaling. The therapeutic efficacy of miR-654 was tested in the pristine-induced lupus mouse model. We further measured miR-654 expression and analyzed its relationship with MIF expression in SLE patients.Results: The serum MIF level was increased in SLE patients (p < 0.001) and positively correlated with the SLEDAI score . MiR-654 inhibited MIF and downstream inflammatory cytokine production by selectively inhibiting the phosphorylation of ERK and AKT. Activation of miR-654 reduced IL-1β, IL-6, IL-8, and TNF-α production, reduced gomerulonephritis, and decreased MIF, IgG, and C3 expression in murine lupus glomeruli. Furthermore, MIF was negatively correlated with miR-654 expression  in SLE patients.Conclusion: MiR-654 negatively correlated with MIF and disease activity in patients with SLE. MiR-654 inhibits MIF expression via binding to MIF 3'UTR, selectively suppresses the phosphorylation of ERK and AKT, and reduces downstream inflammatory cytokine production. In vivo miR-654 treatment decreases MIF and downstream cytokine production and ameliorates murine lupus nephritis. Systemic lupus erythematosus (SLE) is a prototypic immune complex disease that affects multiple organ systems. The immunologic features of SLE include autoantibodies directed against nuclear components and elevated pro-inflammatory cytokines, such as type I interferon (IFN), IL-1β, IL-6, IL-8, TNF-α, and BAFF , suggestMIF alleles also are linked to disease severity in different populations (lpr Mif−/− mice (lpr or NZB/NZW F1 mice (Macrophage migration inhibitory factor (MIF) and its receptors are expressed in elevated levels by different cell types in SLE , and cirulations , 8. MIF ulations –11. Glom−/− mice , and pha F1 mice . Our gro F1 mice . The preThe microRNAs (miRNAs) are a class of small (21–25 nucleotides) non-coding RNAs that regulate gene expression by promoting mRNA degradation or by translational repression at the 3'UTR of target genes . ExtensiIn this study, We identify miR-654 as a negative regulator of MIF expression that reduces MIF-dependent AKT and ERK phosphorylation and downstream, pro-inflammatory cytokine expression. These findings suggest that targeting miR-654 may be a useful approach for the improvement of lupus nephritis.This research was approved by the Research Ethics Board of Renji Hospital, Shanghai Jiao Tong University School of Medicine. Blood samples from SLE patients and healthy controls (HC) were collected with the informed and written consent of all patients and healthy controls. Patients diagnosed with SLE were based on the American College of Rheumatology 1982 revised criteria for SLE . Among tPeripheral blood mononuclear cells (PBMC) were isolated from whole blood by using Ficoll-Paque gradient centrifugation (Sigma), at 400 g for 20 min at 20°C. The PBMCs then were collected and washed twice with sterile PBS. Jurkat, RAW264.7, and THP-1 cell lines were purchased from the cell bank of Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences. miRNA mimic and miRNA inhibitor were obtained from GenePharma. The sequence of the mimic, inhibitor, and control can be seen in Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. PrimeScript RT Reagent kit (Takara) and SYBR Premix Ex Taq RT-PCR kit (Takara) were used to detect the quantities of mRNA expression. The fold-change of each gene was normalized to GAPDH as the internal control using 2–ΔΔCt method. TaqMan MicroRNA Assay kit (Applied Biosystems) and specific TaqMan probes (Applied Biosystems) were used to detect the expression of miRNAs. RNU6 was used as the internal control for humans, and sno202 was used as the internal control for mice.Cells were harvested in lysis buffer and heated at 95°C for 10 min; the cell lysates then were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore). After blocking with 8% skim milk, the membranes were incubated with primary antibodies directed against phosphorylated ERK (Thr202/Tyr204), ERK, phosphorylated Jun (Ser73), Jun, AKT, PI3K, GAPDH, tubulin , MIF  at 4° overnight. After addition of HRP-conjugated secondary antibody , the membranes were washed and the protein bands visualized by chemiluminescence detection kit (Thermo) and quantified with density-analysis software. Anti-tubulin antibody was used as a sample loading control.Protein levels of MIF in sera, MIF, IL-1β, IL-6, IL-8, TNF-α in cell supernatants, and mouse sera MIF, IL-1β, IL-6, IL-8, TNF-α were measured with enzyme-linked immunosorbent assay (ELISA) kit (R&D). Antinuclear antibodies (ANA), anti-dsDNA antibodies and C3 in mouse sera were measured by ELISA kits . Urine protein expression levels were examined by ELISA kit (Sigma-Aldrich). All ELISAs were carried out according to the manufacturer's instruction.A psi-check2 vector containing the MIF 3'UTR sequence was constructed. As the MIF 3'UTR sequence is very short, the 3'UTR sequence was synthesized directly. The sense primer sequence is:CTCGAGGAGCCGCAGGGACCCACGCTGTCTGCGCTGGCTCCACCCGGGAACCCGCCGCACGCTGTGTTCTAGGCCCGCCCACCCCAACCTTCTGGTGGGGAGAAATAAACGGTTTAGAGACT.and the anti-sense primer sequence is:GCGGCCGCAGTCTCTAAACCGTTTATTTCTCCCCACCAGAAGGTTGGGGTGGGCGGGCCTAGAACACAGCGTGCGGCGGGTTCCCGGGTGGAGCCAGCGCAGACAGCGTGGGTCCCTGCGGCTC.5 cells/well in a 24-well plate 1 day before transfection and then transfected with a mixture of 100 ng psi-check2 vector, psi-check2 MIF 3'UTR vector, or psi-check2 MIF 3'UTR-mutant vector together with 100 nM miR mimic, inhibitor, or negative control using Lipofectamine 3000 (Invitrogen). The cells were harvested 24 h later and the Dual-Luciferase Reporter Assay System (Promega) was used to detect the fluorescence activity. Renilla luciferase was used to normalize the firefly luciferase.The psi-check2 vector was digested with Not I and Xho I for 1 h at 37° overnight and the digested vector was used to assemble the MIF 3'UTR sequence. The psi-check2 MIF 3'UTR-mutant vector was constructed by using the TaKaRa MutanBEST Kit (Takara). For the Luciferase assay, 293T cells were seeded at 2 × 10ad libitum feeding at the Shanghai Renji Hospital. Female mice at 8–10 weeks were used for experiments. The lupus mouse model was induced by intraperitoneal administration of 0.5 ml pristine (Sigma) one time  and were maintained in the animal facility with one time . Each grKidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm thick sections and stained with periodic acid–Schiff reagent. Assessments of renal lesions by the activity score described for humans , which graded from 0 to 3 . Tissues were scored by two kidney pathologists in a blinded manner.t-test or the Mann-Whitney U-test as appropriate. All the correlation analyses were performed by Spearman correlation. A p < 0.05 was considered statistically significant. All analyses were performed using Graph Pad Prism Version 6.Categorical data were expressed by counts or percentages, and compared between different groups using the chi-squared test or continuity correction as appropriate. The continuous variables were presented as the mean (standard deviation), and were analyzed by Student's The clinic characteristics of SLE patients and healthy were listed in P < 0.001, P < 0.001, P < 0.001) was observed, in agreement with prior reports  , while there was a negatively relationship between MTX and MIF expression  mRNA was more highly expressed in PBMCs from SLE patients than from healthy controls  . We alsoMIF 3' UTR as predicted by sequence-based bioinformatics analysis. The tools included miRBase, miRecords, PMRD, miRWalk, TarBase, TargetScan, microRNA. org, miRanda, and RNAhybrid. miR-654 was among the 10 miRNAs predicted to target the MIF 3' UTR. The binding sites can be seen in MIF (psi-chek2-MIF 3'UTR). The psi-chek2-MIF 3'UTR vector, different miRNA mimics, and negative control (NC) were transfected into 293T cells, respectively. Using luciferase reporter assay, we screened candidate miRNAs for inhibition of the chemiluminescence in 293T cells transfected with psi-chek2-MIF 3'UTR compared with negative control (http://www.genecards.org), we measured MIF protein level by ELISA in Jurkat T cells transfected with five candidate miRNA mimics or negative control and found three miRNAs that inhibit the MIF protein expression (The above data clearly show MIF is connected to SLE disease activity. MiRNAs have been shown to be involved in the pathogenesis of SLE . Therefo control . Based opression . In our pression . In thisMIF 3'UTR vector (psi-chek2- MIF 3'UTR-Mutant) that we transfected into 293T cells by luciferase reporter assay. We found that miR-654 mimics did not affect the chemiluminescence of 293T cells transfected with psi-chek2-MIF 3'UTR-Mutant fragments in which the 7 base target sites were mutated . miR-654 expression was significantly increased after transfection with miR-65mimic (Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory mediator that upregulates the expression of IL-1β, IL-6, IL-8, TNF-α, et al , 24–27. -65mimic . MIF exp-65mimic . The exp-65mimic .Macrophage migration inhibitory factor (MIF) binds to its transmembrane receptor CD74 to activate CD44, or binds to other receptors, CXCR4 and CXCR2, and initiates a signaling cascade mediated by phosphorylation of ERK, Jun, and PI3K/AKT. These cytosolic elements induce expression of pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNFα. We examined whether miR-654 influences the expression of phosphorylation ERK, Jun, or PI3K/AKT, which may explain the molecular mechanism of miR-654. Tyrosine phosphorylation of ERK and AKT were significantly decreased in MIF stimulated RAW264.7 macrophages after overexpression of the miR-654 mimic, while total ERK, pJun, Jun, AKT, and PI3K protein levels were not significantly affected . As MIF Since exogenous MIF was not affected by miR-654, how miR-654 can affect exogenous MIF signal transduction? We examined the expression of MIF receptors by qPCR, and found that among the four classical MIF receptors: CD74, CD44, CXCR2, CXCR4. Only the expression of CD44 is significantly decreased by overexpression of miR-654 . Furtherin vivo, miR-654 mimic or NC were mixed with Lipofectamine® RNAiMAX and injected into lupus mice induced with pristine. Mice were sacrificed 6 months later to analyze renal pathology and serum autoantibodies, complement, and cytokines (P < 0.05). miR-654 expression was significantly increased in the kidney in the miR-654 group(P < 0.05, As shown above, miR-654 can suppress the expression of MIF and inflammatory cytokines. In order to investigate the biological relevance of miR-654 ytokines . PristinP < 0.05, P < 0.05, lpr lupus models (In order to better understand the role of miR-654 in the pathogenesis of SLE, we treated mice with pristine 4 weeks before treating them with the miR-654 inhibitor . Five mos models .P < 0.05, Sera MIF expression was reduced in miR-654 treated group mouse (P < 0.05, The expression of serum MIF was significantly increased in the miR-654 inhibitor treated mouse when compared with the NC group (P < 0.0001, P < 0.001, P < 0.01, P < 0.05, Given that miR-654 can regulate the expression of MIF and no study has reported the function or expression of miR-654 in SLE, we examined the expression of miR-654 in SLE patients and analyzed its relationship with clinical characteristics. MiR-654 expression was significantly reduced in SLE patients (MIF alleles and renal injury (Renal involvement is very common in SLE and severe lupus nephritis is a leading cause of morbidity and ultimate mortality. Autoantibody and immune complex deposition in the microvasculature of the kidney creates a proinflammatory microenvironment that leads to irreversible glomerular injury, tubular inflammation, and loss of function . MIF is l injury , the amel injury , 13, andl injury , all obsl injury . NeverthMorand et al. have reported that MIF expression is higher in Asian than other ethnic SLE cohorts . All SLEPrevious studies have demonstrated that miRNAs play important role in the pathogenesis of SLE. For example, miR-146a activates the type I IFN pathway in human lupus by targeting the key signal transducer Stat1 . miR-150MIF expressers (In the pristine-induced mouse model of SLE, the experimental administration of miR-654 did not influence circulating ANA and anti-dsDNA antibody levels, suggesting little impact on dysregulation of the adaptive response. Taken together, these results indicate that miR-654 may play a pivotal role in the progression of lupus nephritis by regulating pathologic MIF overexpression. These data also imply that miR-654 may have potential therapeutic application in lupus nephritis, particularly in individuals who are with high genotypic pressers . Importapressers .The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All animal experiments complied with the relevant institutional guidelines of animal ethics from Renji Hospital, Shanghai Jiao Tong University, School of Medicine.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C. elegans revealed organism-specific, development-dependent and environment-driven metabolic features. We showed for the first time the presence of a series of sphingoid bases in C. elegans sphingolipid profiles, although only C17-sphingoid base is used for generating complex SLs. Moreover, we successfully resolved growth-, temperature- and nutrition-dependent SL profiles at both individual metabolite-level and network-level. Sphingolipidomic analysis uncovered significant SL composition changes throughout development, with SMs/GluCers ratios dramatically increasing from larva to adult stage whereas total sphingolipid levels exhibiting opposing trends. We also identified a temperature-dependent alteration in SMs/GluCers ratios, suggesting an organism-specific strategy for environmental adaptation. Finally, we found serine-biased GluCer increases between serine- versus alanine-supplemented worms. Our study builds a “reference” resource for future SL analysis in the worm, provides insights into natural variability and plasticity of eukaryotic multicellular sphingolipid composition and is highly valuable for investigating their functional significance.Sphingolipids (SLs) serve as structural and signaling molecules in regulating various cellular events and growth. Given that SLs contain various bioactive species possessing distinct roles, quantitative analysis of sphingolipidome is essential for elucidating their differential requirement during development. Herein we developed a comprehensive sphingolipidomic profiling approach using liquid chromatography-mass spectrometry coupled with multiple reaction monitoring mode (LC-MS-MRM). SL profiling of SLs are involved in a wide array of cellular processes, such as protein modification, intracellular trafficking, proliferation, differentiation, apoptosis, angiogenesis, and stress response  Saccharomyces cerevisiae and Drosophila are the two organisms whose lipidomics has been studied at whole organism level, with tremendous advance in quantification of some of the SL classes , as well as in identification of the connections between cell behavior and SL alteration The SL metabolic pathway reveals a complex and often circular network of chemical reactions catalyzed by well conserved enzymes producing a multitude of diverse intermediates that interconnect C. elegans as a model system, significant progress has been made in functional and structural characterization of various SL species in the past decades. Specifically, ceramides are involved in IR-induced germ cell apoptosis Drosophila and mammalian, there is only one single sphingoid base in C. elegans with a unique C17 branched-chain fatty acid (FA) C. elegans contains all functionally distinct sphingoid derivatives, including simple SLs ; N-acyl-derivatives (e.g. ceramide [Cer] and ceramide 1-phosphate [C1P]); and complex SLs (e.g. sphingomyelin [SM] and glucosylceramide [GluCer]). This simplicity allows us to use C. elegans for the analysis of sphingolipid homeostasis under various conditions, a prerequisite for any functional analysis of specific SLs.Using  C. elegans as a model organism. The regulation of SL composition during growth and development (with entirely different requirements for SL biosynthesis) C. elegans SL profiles under these specific growth states. Importantly, this analysis provides a baseline characterization of the homeostatic biochemical network and establishes LC-MS-MRM based sphingolipidomics as a powerful platform for complementing genetics studies in worms.Here we conducted a systematic and comprehensive investigation on sphingolipidomics usingThe Ceramide/Sphingoid Internal Standard Mixture containing 25uM each of 10 compounds, including C17-sphinganine (C17-Sa), C17-sphingosine (C17-So), C17-sphingosine-1-phosphate (C17-So1P), C17-sphinganine-1-phosphate (C17-Sa1P), C12-ceramide (C12-Cer), C25-ceramide (C25-Cer), C12-ceramide-1-phosphate (C12-C1P), C12-SM, C12-glucosylceramide (GluCer), C12-lactosyl (β)-ceramide, was obtained from Avanti Polar Lipids . C18-1-deoxy-sphinganine (C18-DOSa), C18-1-deoxymethyl-sphinganine (C18-DOMSa) were also purchased from Avanti Polar Lipids, and dissolved as 25uM in DMSO and used as standard for detecting and quantifying deoxy-sphingoid bases. Other reagents used were as follows: methanol , acetonitrile , 2-propanol, ammonium acetate and potassium hydroxide , formic acid , glacial acetic acid . Water was obtained from a Milli-Q Water Purification System from Millipore .C. elegans  worms were cultured and maintained at 22°C on standard nematode growth media (NGM) seeded with OP50 bacteria unless indicated otherwise. Worm synchronization was carried out using a standard protocol as previously described Wild-type C. elegans was carried out according to a modified Bligh and Dyer's method 3), vortexed for 1 min, and incubated in a water bath  at 48°C for 24 hrs. The supernatant was collected and the solvent was evaporated by nitrogen blowing at room temperature. 75 μL of 1 M potassium hydroxide (in MeOH) was added into the residues and the mixture was sonicated for 5 min. After an incubation at 37°C for 2 hrs, 3 μL glacial acetic acid was added for neutralization followed by a phase separation generated by adding 1 mL CHCl3 and 2 mL water. Following a centrifugation at 2,500 rpm for 10 min, the upper layer (water layer) was carefully removed and discarded; and the chloroform layer was dried under vacuum and reconstituted with CHCl3-MeOH (2:1). The samples were centrifuged several times to further remove impurities before LC-MS analysis.The worm samples were thawed and homogenated firstly by a sonicator , and then freeze-dried  for 24 hrs. The extraction of SLs from The SL separation and analyses were performed using a Waters Acquity ultra-high performance liquid chromatography (UPLC) system in tandem with Waters Xevo TQD triple quadrupole mass spectrometers . The LC-MS system was equipped with a binary solvent manager and an autosampler. Waters Xevo TQ-MS was set in a positive electrospray mode for quantification of SLs coupled with multiple reaction monitoring (MRM). The capillary voltage and the source extractor voltage were set at 3.5 kV and 3 V, respectively, and the cone voltage was 40 V. The source and the desolvatation gas temperatures were maintained at 140°C and 500°C, respectively. The desolvatation and cone gas flows were 500 L/hr and 1 L/hr, respectively. Dwell time and inter-channel delay were set to 20 and 5 ms. Data acquisition, data analysis and instrument control were done by Empower Pro 6.0 , and MS system was controlled by Analyst 1.4.2 software . The standards and samples were separated using the following two gradient elution chromatographic separations.3OH : CH3COOH = 97 : 2 : 1 (v/v) containing 5mM NH4OAC; mobile phase B consisted of CH3OH : CH3COOH = 99 : 1 containing 5mM NH4OAC. The gradient elution was as follows: 0 - 1 min, 100% A; 1 - 4 min, 100 - 0 % A; 4 - 8 min, 100 % B; 8 - 10 min, 100 - 0 % B; 10-12 min, 100% A. The flow rate was set at 0.4 ml/min.Method 1  - A waters ACQUITY UPLC BEH Amide column (1.7 μm 2.1×100mm) with an in-line filter was maintained at 45°C. Mobile phase A consisted of CAN : CHMethod 2  - A Waters ACQUITY UPLC BEH C18 column (1.7 μm 2.1×100mm) with an in-line filter was maintained at 40°C. Mobile phase A consisted of 0.1% formic acid in ACN/water ; mobile phase B consisted of 0.1% formic acid in ACN/2-propanol . The gradient elution was as follows: 0-1 min, 70% A; 1-2.5 min, 60-30% A; 2.5-4 min, 30-20% A; 4-5 min, 20% A; 5-6.5 min, 20-10 % A; 6.5-6.6 min, 10-0 % A; 6.6-8 min, 100% B. The flow rate was set at 0.25 ml/min.Data analysis including peak smoothing and integration of areas under the curves for each ions was performed by Masslynx software (Waters). The semi-quantitation of individual metabolites was normalized with the respective internal standards, and calculated with the formula as follows.p<0.05) and T-test . Multivariate and univariate analyses and partial least squares-discriminate analysis (PLS-DA) were used to identify features that differed markedly between different experimental groups, and performed based on the concentrations of SLs (>50 variables) by MetaboAnalyst 4.0 The level of statistical significance was set by one-way ANOVA assay , temperatures  and nutritional conditions . Targeted SL species include sphingoid bases, N-acyl-derivatives and complex SLs according to the SL metabolic pathway  for each particular SL subclass using internal standard to achieve the best sensitivity and consistency. Comparative analysis of the ionization efficiency and separation precision with normal phase (NP)- and reverse phase (RP)-LC showed that S1P detection was significantly enhanced with RP-LC , degree of saturation and position of the double bonds, presence of a hydroxyl group at position 4 and branching of the alkyl chain have been reported C. elegans, Cers are 5-10 times more abundant than free sphingoid bases, yet 50 times less than complex SLs, and within the N-acyl derivative class, the abundance of each subclass is in an order of Cer>CerOH>DHCer>DHCerOH. Of note, regardless of detection of various sphingoid bases, DHCers and Cers derived from sphingoid bases other than C17, such as Cers (d18:1), Cers (d19:1), DHCer(d18:0) and DHCer(d19:0), or from DSBs, such as Cers (m17:0) and Cers (m19:0), were not found in C. elegans (data not shown).As the N-acyl derivatives of sphingoid bases, DHCer and Cer consist of a sphinganine and sphingosine backbone, respectively, linked with a FA through an amide bond. In line with previous reports C. elegans, and C22-GluCer showed the highest abundance  to the ceramide unit. GluCers d17:1/C21:0-26:0) broadly existed in 1:0-26:0 nce Fig. . SM consant Fig. .C. elegans sphingolipidome by monitoring lipid species constituting the 9 major SL classes in C. elegans. We excluded from our further analysis certain low abundant SL class and species that, for technical reasons, could not be robustly quantified . Considering the low diversity of elements in SLs  as well as the long chains built by C-C bonds, the possibilities of multiple analytes possessing the same molecular formulas but different structures or atom arrangement exist. It is worth to note that the monomethyl-branched chain feature of the sphingoid bases and the precise location of double bonds and hydroxylations cannot be derived from our analytical approach.In summary, the sphingolipidomics platform we developed featured a broad dynamic quantification range covering 3-4 orders of magnitude and a detection limit in the low ng/ml range. We estimated that the quantitative analysis covered 90% of the We next applied our targeted workflow to systematically measure SL composition variations during worm development, or when grown at different temperatures and under different nutritional conditions. To assess the main metabolic changes, we focused on a set of metabolic intermediates that show relatively high abundance and compared the normalized abundances of SL classes in worms under various conditions. Partial least square discriminant analysis (PLS-DA) of LC-MS/MS-based sphingolipidomics showed clear clustering of the metabolic profiles between different sample groups Fig. . MultivaHierarchical clustering based on metabolite abundance revealed striking differences in SL profile of worms at different developmental stages Fig. . SL compWe next compared the SL compositions of L2- and adult-stage animals in more detail. Hierarchical clustering analysis of the two stages show a significant increase of majority of SL species from L2 to adult stage Fig. A, wherea C. elegans at 16°C, 20°C and 22°C. As shown in Fig. The effect of temperature on the SL composition was examined by growingRemarkably, different classes of structurally more complex SLs exhibit different responses upon temperature changes. Cers seemed to increase with temperature rising, although not in a statistically significant manner probably due to low abundance. Most of GluCers were significantly more abundant at 22°C with little abundance changes between 16-20°C whereas levels of some SMs were gradually increased as the temperature drops, and consequently the ratio of SMs versus GluCers was much higher at 16°C than that at 20°C and 22°C Fig. A-B, D-E.C. elegans by growing C. elegans on NGM plates supplemented with alanine or serine . Supplementation of alanine or serine led to the accumulation of majority of the detected SLs to a similar extent, including sphingoid bases and complex SLs  we did not try to resolve the FA chain and monosaccharide configuration  and the related conclusions were drawn based on the previous studies; 2) some mass-to-charge (m/z) values remain conflicted with anticipated isobaric overlap ; 3) the amounts detected should not be considered as absolute concentration due to the structural difference between C. elegans-specific sphingolipid species and standards.Investigating the intrinsic and extrinsic variability of SLs induced by different growth conditions in multicellular organisms is essential for better understanding biological roles and significance of SLs. This study established the LC-MS-MRM methodology as a valuable tool for molecular characterization of the C. elegans, as a model organism for biological research in many fields including genetics, development and aging, has a well-defined developmental pattern. Once fertilized, C. elegans proceeds through early embryogenesis, four larval stages (L1-L4) - each separated by molting, and become a reproductive adult within 3-4 days. When unfavorable conditions  persist, C. elegans undergoes growth arrest, either as an L1 larva or as a specialized L2 diapause form termed the dauer larva S. aureus maintains membrane structure and fluidity through reducing the content of branched chain FAs when grown at a higher temperature Vibrio strain regulates membrane fluidity by carrying out cis to trans isomerization of unsaturated FAs in response to temperature DrosophilaC. elegans. It would be interesting to monitor FA desaturation in phospholipids of worms grown at different temperatures. On the other hand, given that SLs mainly contain saturated FAs, changing SL composition and FA chain length of SLs may serve as the supplementary mechanism for worms to adapt to temperature changes.It is known that lipid compositions are highly dependent on temperature C. elegans is thought to contain rafts C. elegans in greater abundance to replace the structural functions ascribed to cholesterol in other animal cell membranes and they may take over the regulation of membrane fluidity and permeability as significant changes in the ratio of shorter SMs(FA≤C22) versus longer SMs(FA>C22) were detected responding to growth temperature Some lipid mixtures form membranes containing submicroscopic ordered lipid domains (rafts) that serve as platforms for signal transduction or protein sorting Drosophila and mouse L-Serine is required to synthesize membrane lipids such as phosphatidylserine and SLs. Profound metabolic and physiological changes have been observed through manipulating serine content in various organisms. Deprivation of external L-serine through genetic depletion of L-serine biosynthetic enzyme leads to the generation of DSBs, including DoSa in mouse embryonic fibroblasts, which can be further suppressed or potentiated by supplementing exogeneous L-serine or increasing the ratio of L-serine to L-alanine, respectively Supplementary figures and tables.Click here for additional data file.


Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC.aimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure.The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCThe combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.The online version of this article (10.1186/s13007-018-0351-2) contains supplementary material, which is available to authorized users. Important traits of crop plants have successfully been manipulated by selection of mutants , by ectohttp://www.mirbase.org/) only a small number have been functionally characterised. Moreover, while most miRNA are processed from regular non-coding miRNA-genes, recently, protein-coding genes with introns containing potential miRNA sequences have been identified both in mammals and plants. For instance, the Arabidopsis thaliana genome contains 37 protein coding genes with intronic miRNAs (imiRNAs) and the rice genome contains 181 protein coding genes with imiRNAs [C. elegans, zebra fish, and chicken [MicroRNAs (miRNA) are short 19-22nt) non coding RNAs that can silence the expression of specific target genes and natural miRNAs form an integral part of developmental decisions in plants -22nt non. From al imiRNAs . At pres imiRNAs . In such chicken . It has  chicken , 15. The chicken . How imi chicken . MoreoveaimiR-319a, ffgLUCaimiR-LUC and ffgLUCaimiR-PHYB), designed to report on both protein and miRNA function. For overexpression, the firefly Luciferase gene with a single intron (ffgLUC) was used, which allows for easy monitoring of gene activity and splicing accuracy. As template for the miRNA sequence in the intron, the sequence of the natural ath-miR319a was used [Arabidopsis thaliana PHYB mRNA (AT2G18790). Initially, insertion of the miRNA into the LUC intron resulted in a loss of LUC activity, indicating incorrect splicing of the intron from the LUC pre-mRNA. However, after adjusting the miRNA position within the intron, the transgene showed normal LUC activity when expressed in plants, indicating accurate splicing of the LUC pre-mRNA. Moreover, the aimiRNA targeting TCPs or ffgLUC both were able to suppress target gene expression, indicating effective processing of the aimiRNA from the ffgLUC intron. The concept of a transgene containing an aimiRNA could be useful for simultaneous manipulation of several gene activities, which could be an important tool for plant biotechnology.Here, we tested whether a protein coding transgene can be effectively expressed in plants, while also producing a functional miRNA. The feasibility and requirements for such dual gene functionality were determined using three gene construct  was used for stable transformation. The Arabidopsis phyB-9 T-DNA insertion mutant (#CS6217) was obtained from the NASC stock collection. Plants were grown on rock-wool in a growth chamber at 12hL/12hD at 22 °C on half strength Hoagland-nutrient solution.AthPHYB (AT2G18790) were selected using WMD3-Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). The IDT RNAi design tools (Integrated DNA Technologies) was used to design the amiRNA nucleotide sequence targeting the luciferase mRNA (5′-TAGAACTGCCTGCGTCAGATT-3′). Pre-microRNA 319a was amplified directly from A. thaliana genomic DNA using primers (CAAACACACGCTCGGACGCAT-F and CATGGCGATGCCTTAAATAAAG-R). The aimiRNA sequences were amplified from pre-miRNA319a using specific primers which added EcoR V and EcoR I restriction sites for cloning into the intron of ffgLUC (GATATCAGAGAGCTTCCTTGAGTCCATTCAC-F and GAATTCAGGGAGCTCCCTTCAGTCCAATC-R). For amplification of the aimiR-LUC the TCP target sequence in the primers was replaced by the selected LUC target sequence (GATATCTATAACTGCCTGCCTCAGATAAGGTCGTGATATGATTCA-F and GAATTCTAGAACTGCCTGCGTCAGATTAAAGAGAATCAATGATCCA-R). For the amplification of the aimiR-PHYB the TCP target sequence in the primers was replaced by the selected PHYB target sequence (GATATCTAGCTGTAAACCGTAAGGCTCAGGTCGTGATATGATTCA-F and GAATTCTAACTGTAAACCGAAAGGCTGAAAGAGAATCAATGATCCA-R). To generate of ffgLUC delaimiR319a construct the first exon plus 10 nucleotides from 5′ site of intron was amplified by using primer which introduce Nco I at start codon and EcoRV site in intron (CCATGGAAGACGCCAAAAAC-F and GATATCAGAAACTTACGTAATGTTCACCTCG-R). The second exon plus 61 base pair from 3′ site of the intron was amplified using primers which introduce an EcoR I site at the end of the intron sequence and an Not I site after the stop codon (GAATTCAACTTTTCTAATATATGACCAAAATTTGTT-F and GCGGCCGCTTACAATTTGGACTTTCCGCCCTT-R). To generate of ffgLUCimiR319a ffgLUCaimiR-LUC and ffgLUCaimiR-PHYB constructs, the first exon plus 33 nucleotides from the 5′ end of the intron was amplified by using primer pairs introducing an Nco I at the ATG start codon and an EcoR V site at the end of the intron sequence (CCATGGAAGACGCCAAAAAC-F and GATATCTACTAATTAATGATAATTATT-R). The second exon of ffgLUC was amplified from 135 base pairs from 3′ splice site to after the stop codon, introducing an EcoR I site at in the intron and Not I site after the stop codon, using the primer pairs (GAATTCGTAATATAATATTTCAAATATTTTTTTCAAAATAA-F and GCGGCCGCTTACAATTTGGACTTTCCGCCCTT-R). The resulting PCR products were digested with EcoR I and EcoR V and the amiRNAs product was ligated into the ffgLUC intron. The ffgLUC was amplified with primers introducing an Nco I site at the ATG and Not I site after the stop codon (CCATGGAAGACGCCAAAAAC-F and CGGCCGCTTACAATTTGGACTTTCCGCCCTT-R). The ffgLUC, ffgLUCimiR319a, ffgLUCaimiR-LUC or ffgLUCaimiR-PHYB constructs were subsequently ligated into the Nco I/Not I sites of pIVA2.1 entry vector which contained double 35S promoter and RubescoS terminator. To generate the binary vector, all pIVA2.1-based vectors were cloned into the pKGW_RedSeed vector [Artificial microRNAs constructs were created using ath-miR319a backbone as described by Liang . The prid vector  through trans the artificial microRNAs 2×35S::amiR-LUC and 2×35S::amiR-PHYB constructs were generated using primer sets which replace the TCP target sequence in miR-319a with target sequences for LUC or PHYB respectively (LUC: CCATGGTATAACTGCCTGCCTCAGATAAGGTCGTGATATGATTCA-F and GCGGCCGCTAGAACTGCCTGCGTCAGATTAAAGAGAATCAATGATCCA-R or PHYB: CCATGGTAGCTGTAAACCGTAAGGCTCAGGTCGTGATATGATTCA-F and GCGGCCGCTAACTGTAAACCGAAAGGCTGAAAGAGAATCAATGATCCA-R). The PCR products were cloned into pIVA2.1 entry vector which was subsequently used for recombination into the pKGW_RedSeed vector. All destination vectors were subsequently transformed to Agrobacterium tumefaciens (AGL0).For confirmation of LUC or PHYB silencing in Agrobacterium tumefaciens was used for plant transformation using the floral dip method as described [0 seeds were identified by DsRed pigmentation of the seed coat. For germination seeds were plated on 3% water agar plates and cold-treated for 5 days at 4 °C after which plates were incubated in growth chambers in the light at room temperature. After 3 days, germinated seedlings were transferred to soil or rock wool for plant growth.escribed . TransgeN. benthamiana using agrobacterium strains carrying the different expression vectors (or empty vector) was done as described by Wang [Agro-infiltration in  by Wang .Arabidopsis thaliana plants were sprayed with 1 mM D-luciferin  24 h and 1 h before imaging with an (− 80 °C) air-cooled CCD Pixis 1024B camera system  equipped with a 35 mm, 1:1.4 Nikkon SLR camera lens  fitted with a DT Green filter ring  to block chlorophyll fluorescence. Exposure time is as indicated.For LUC activity measurements in stable transformed N.benthamiana leaves were harvested 4 days post agro-infiltration. Leaves were sprayed with 1 mM D-luciferin at 24 and 1 h before imaging (5 min exposure time). Relative luminescence from LUC activity was analysed in Image J , using background subtraction. For each treatment LUC activity in leaves from 6 to 8 independent plants was quantified.For transient assays, For hypocotyl length measurement, seeds were surface sterilized and imbibed on 0.25% water agar plates at 4 °C in the dark, after which plates were transferred to a Red LED light box (50 uMol) at 22 °C. Seedlings were flattened at 5 days after transfer, and hypocotyl length was determined from photograph in Image J . At least 20 seedlings were scored from each genotype.3 generation plants were grown for 4 weeks. The RNA was extracted from rosette leaves from WT (Col-0), ffgLUCdelaimiR-319a, ffgLUCaimiR-319a or ffgLUCaimiR-PHYB transformants using InviTrap Spin Plant RNA mini Kit , following manufacturer’s instructions. Purified total RNAs were subjected to TURBO DNA-free™ DNase  treatment to avoid with contaminated genomic DNA. For reverse transcription the iScrip II mix reagent was used that included 10 mM oligo (dT) primer according to the manufacturer’s instruction . The primers listed in Additional file A. thaliana ACTIN1 was used as reference. RNA analysis from transient assays in N. benthamiana were carried out on RNA isolated from three pooled agro-infiltrated leaves, in triple biological replicates, using N.benthamiana UBI3 as reference genes. The Ct method (2 − ΔΔCt) was used to analysis the differences in mRNA values (http://www.bio-rad.com/). All expression constructs used in the transient assays contain a 35S::DsRed marker gene and quantification of the DsRED gene expression in the transient assays was used to confirm similar transformation frequencies in the different agro-infiltration treatments.For RNA analysis, TdelaimiR-319a, ffgLUCaimiR-319a, and ffgLUCaimiR-PHYB plants and immediately ground in liquid nitrogen with a mortar and pestle. Approximately 100 mg ground leaf tissue was used to small RNA extraction. The extraction of small RNAs were performed by using Prima microRNA Isolation Kit  according to the manufacturer’s protocol. The small RNA purity and concentration was measured by NanoDrop spectrophotometer .Detection of specific small RNAs was by the step-loop PCR method as described by Varkonyi-Gasic . BrieflyThe specific RT primers were used for miR319a and amiR-PHYB in stem-loop RT reaction. Reverse transcription reaction were performed according to Varkonyi-Gasic et al. . Forwardt test (P < 0.001).Comparison of means was analysed for statistical significance with a 2-sample Arabidopsis TCP transcription factor family, was used [To determine whether a functional microRNA can be efficiently generated from an intron of a transgene, without affecting accuracy of intron splicing, both accuracy and efficiency of transgene splicing and efficiency of silencing by the aimiRNA need to be monitored. To monitor transgene splicing the firefly luciferase (ffgLUC) reporter gene with an intron was used  see Fig. To studwas used . When thwas used .Fig. 1StdelaimiR-319a  gene expression was affected by ffgLUCdelaimiR-319a as the AthTCP target sequence of aimiR-319a shows substantial overlap with sequences in NbTCP4. Results show that NbTCP4 expression level was reduced in leaves expressing ffgLUCdelaimiR-319a compared to the control leaves expressing ffgLUC  as described for plants expressing 2×35S::miR319a [AthTCP2, AthTCP3, AthTCP4 indicated that their expression was reduced by ~ 90% in the ffgLUCdelaimiR-319a plants  of the same age . Both mature aimiR-LUC and LUC mRNA are produced from the same pre-mRNA and silencing of LUC activity provides information on the relative efficiency of the two maturation processes (mRNA vs miRNA). In the transient expression assays, LUC activity in N. benthamiana leaves expressing ffgLUCaimiR-LUC showed no significant reduction compared with leaves expressing ffgLUC  expression construct was co-infiltrated with a 2×35S::amiR-LUC expression construct. This showed that also 2×35S::amiR-LUC is not capable of silencing transiently expressed LUC . The aimiR-LUC is based on the sequence and structure of the native miR319a precursor, but the 21 base-pair sequences targeting TCPs are replaced by 21 base-pairs targeting luciferase mRNA  the LUC activity was reduced by 69–53% compared to the original ffcLUC line   and 10 independent transformed T1 plants were grown. From these, eight transformants with a single copy transgene insertion were selected for further analysis. On average, the LUC activity in these eight lines was comparable with that of eight independent transformants expressing ffgLUC . All together, these results suggest that the mature amiR-PHYB derived from ffgLUCaimiR-PHYB is not functional in silencing PHYB expression.After stable transformation of LUC Fig. b. This anes Fig. c. The siIntron-derived miRNAs (imiRNAs) are an alternative source for miRNAs in mammals and plants , 28–31. delaimiR-319a resulted in spatial constraints because of simultaneous assembly of spliceosome and miRNA-processing protein complexes. In contrast, the aimiRNA placed at 55 bp from the 5′-splice site resulted in efficient maturation of the LUC mRNA, resulting in similar LUC activity for ffgLUCaimiRNA-319a and ffgLUC in transient expression as well as stable transformants . Figure S2. RT-PCR products sequence from ffgLUCdelaimiR-319a transgenic plant. Figure S3. A similar transformation efficiency were confirmed by quantify the DsRED gene expression in transient assay samples. The quantification data is normalized against the N. benthamiana reference with UBI3 as internal control. Error bars represent standard error. Figure S4. Confirmation of mature aimiR-319a expression in stem-loop RT-PCR assay. Illustration of RT-PCR method for amplification of mature microRNA (A). Gel electrophoresis results from stem-loop RT (B). ffgLUC plant used as a positive control for endogenous mature miR319a. A mature microRNA specific forward primer and universal reverse primer were used for PCR amplification. Mature microRNA products were obtained using 25 cycling of RT-PCR and analysed on 4% agarose gel in 1xTAE. Predicted products were compared with 50 bp DNA ladder. Figure S5. PHYB silencing phenotype under constant cR. Table S1. Stable transformation of Arabidopsis WT or Arabidopsis line ff-gLUC-1 with the different expression constructs. NA not applicable, *very low LUC activity; **low LUC activity. Table S2. List of primers were used in this work.