In eukaryotes, the RNase-Ⅲ Dicer often produces length/sequence microRNA （miRNA） variants, called ＂isomiRs＂, owing to intrinsic structural/sequence determinants of the miRNA precursors （pre-miRNAs）. In this stu...In eukaryotes, the RNase-Ⅲ Dicer often produces length/sequence microRNA （miRNA） variants, called ＂isomiRs＂, owing to intrinsic structural/sequence determinants of the miRNA precursors （pre-miRNAs）. In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 （AGO1）. We identified a motif conserved among plant premiR168 orthologs, which enables flexible internal basepairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via ＂transitive RNAi＂, a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGOl-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of premiRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.展开更多
To counteract the immune system in parasitic hosts,some viruses encode proteins to suppress the RNA interference(RNAi)effect.In this report,we established two RNAi systems to be easily observed with strong and obvious...To counteract the immune system in parasitic hosts,some viruses encode proteins to suppress the RNA interference(RNAi)effect.In this report,we established two RNAi systems to be easily observed with strong and obvious effect.The function of the P19 of tomato bushy stunt virus,which suppresses RNAi in mammal cells,was then studied using these two systems.Short hairpin RNAs targeting green fluorescence protein(pshRNA-GFP)and firefly luciferase(pshRNA-luc)were designed and inserted into a eukaryotic transcriptional vector pTZU6+1,respectively.The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19.The GFP expression level was assayed by fluorescence microscopy,Western blotting and RT-PCR.The luciferase expression level was analyzed by the dual-luciferase assay system.pshRNA designed in this study down-regulated the target gene specifically and efficiently,with a decrease of expression of both genes of about 70%,respectively.When P19 was introduced into the RNAi systems,the expression of both GFP and the luciferase were mostly recovered compared with the control groups.The RNAi systems of GFP and luciferase were constructed successfully,demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.展开更多
While there is mounting evidence that interleukin （IL）-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathologica...While there is mounting evidence that interleukin （IL）-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathological processes. IL-23 is a heterodimer composed of p19 and p40, the latter being shared with IL-12. We previously reported that prostaglandin （PG） E2 promotes CD40-mediated induction of 1123a （p19） expression through its E receptor subtype 4 （EP4） receptor in splenic dendritic cells （DCs）. Here, we have analyzed signaling pathways regulating 1123a induction in the cross talk between EP4 and CD40 in bone marrow-derived DCs. We found that PGE2 synergistically induced 1123a transcription with CD40 signaling. An EP4 agonist, but not agonists of EP1, EP2, or EP3, reproduced this action. Stimulation of CD40 with an agonist antibody evoked biphasic induction of 1123a expression, with the early phase peaking at 1 h and the late phase peaking at 12 h and lasting up to 36 h after stimulation, whereas induction by lipopolysaccharide or tumor necrosis factor-α was transient. The early phase induction by CD40 stimulation was absent in DCs derived from Nfkbl-deficient mice, and the late phase induction was eliminated by RNA interference of nuclear factor-kappa B （NF-κB） p100 subunit. Further, cAMP response element-binding protein （CREB） depletion completely eliminated the induction of 1123a by CD40 stimulation. The addition of the EP4 agonist amplified the induction in both phases through the cAMP-protein kinase A （PKA） pathway. These results suggest that 1123a expression in DCs is synergistically triggered by the PG E2-EP4-cAMP-PKA pathway and canonical/non-canonical NF-KB pathways and CREB activated by CD40 stimulation.展开更多
The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines(TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcin...The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines(TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid(p Tα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, q T-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.展开更多
Objective： To investigate the changes of cyclin L2（CCNL2） gene mRNA and protein during the differentiation of P19 cells to cardiac myocytes, and to explore the relationship between CCNL2 gene and the differentiatio...Objective： To investigate the changes of cyclin L2（CCNL2） gene mRNA and protein during the differentiation of P19 cells to cardiac myocytes, and to explore the relationship between CCNL2 gene and the differentiation of cardiac myocytes. Methods： P19 cells were cultured with 0.9% DMSO for 18 days. Western blots of cardiac troponin I （cTnI） were used to identify cell differentiation. Total RNA was extracted from P19 cells during the process of differentiation at various time points：pre-differentiation（Day 0）, and Day 1 to Day 18. The expression levels of CCNL2 gene mRNA and protein were evaluated by RT-PCR and Western blot, respectively. Results： After being induced to differentiate by DMSO for 4 days in suspension, spontaneously and rhythmically beating ceils were seen at 8 day, which were cTnI-positive. In P19 cells, both the expression level of CCNL2 gene mRNA and protein were gradually down-regulated. Conclusion： Both the expression of CCNL2 gene and protein were down-regulated during the process of the differentiation of P19 cells into cardiac myocytes, suggesting a possible role for this cyclin in their differentiation.展开更多
基金The NCCR RNA and disease funded by the Swiss National Science Foundation （SNF 51NF40_141735） for access to the Biomolecular NMR spectrometry platform at ETH ZUrich. This work was supported by an IIF Marie Curie fellowship to TI （no. 329029） and a European Research Council （ERC） advanced grant ＂Frontiers of RNAi-Ⅱ＂ to OV （no. 323071）.
文摘In eukaryotes, the RNase-Ⅲ Dicer often produces length/sequence microRNA （miRNA） variants, called ＂isomiRs＂, owing to intrinsic structural/sequence determinants of the miRNA precursors （pre-miRNAs）. In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 （AGO1）. We identified a motif conserved among plant premiR168 orthologs, which enables flexible internal basepairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via ＂transitive RNAi＂, a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGOl-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of premiRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.
基金National Natural Science Foundation of China (No.30500428)Chongqing Science and Technology Commission (2006BA5021)
文摘To counteract the immune system in parasitic hosts,some viruses encode proteins to suppress the RNA interference(RNAi)effect.In this report,we established two RNAi systems to be easily observed with strong and obvious effect.The function of the P19 of tomato bushy stunt virus,which suppresses RNAi in mammal cells,was then studied using these two systems.Short hairpin RNAs targeting green fluorescence protein(pshRNA-GFP)and firefly luciferase(pshRNA-luc)were designed and inserted into a eukaryotic transcriptional vector pTZU6+1,respectively.The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19.The GFP expression level was assayed by fluorescence microscopy,Western blotting and RT-PCR.The luciferase expression level was analyzed by the dual-luciferase assay system.pshRNA designed in this study down-regulated the target gene specifically and efficiently,with a decrease of expression of both genes of about 70%,respectively.When P19 was introduced into the RNAi systems,the expression of both GFP and the luciferase were mostly recovered compared with the control groups.The RNAi systems of GFP and luciferase were constructed successfully,demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.
文摘While there is mounting evidence that interleukin （IL）-23-IL-17 axis plays a critical role in the pathogenesis of various autoimmune diseases, much remains to be elucidated on how IL-23 is induced in the pathological processes. IL-23 is a heterodimer composed of p19 and p40, the latter being shared with IL-12. We previously reported that prostaglandin （PG） E2 promotes CD40-mediated induction of 1123a （p19） expression through its E receptor subtype 4 （EP4） receptor in splenic dendritic cells （DCs）. Here, we have analyzed signaling pathways regulating 1123a induction in the cross talk between EP4 and CD40 in bone marrow-derived DCs. We found that PGE2 synergistically induced 1123a transcription with CD40 signaling. An EP4 agonist, but not agonists of EP1, EP2, or EP3, reproduced this action. Stimulation of CD40 with an agonist antibody evoked biphasic induction of 1123a expression, with the early phase peaking at 1 h and the late phase peaking at 12 h and lasting up to 36 h after stimulation, whereas induction by lipopolysaccharide or tumor necrosis factor-α was transient. The early phase induction by CD40 stimulation was absent in DCs derived from Nfkbl-deficient mice, and the late phase induction was eliminated by RNA interference of nuclear factor-kappa B （NF-κB） p100 subunit. Further, cAMP response element-binding protein （CREB） depletion completely eliminated the induction of 1123a by CD40 stimulation. The addition of the EP4 agonist amplified the induction in both phases through the cAMP-protein kinase A （PKA） pathway. These results suggest that 1123a expression in DCs is synergistically triggered by the PG E2-EP4-cAMP-PKA pathway and canonical/non-canonical NF-KB pathways and CREB activated by CD40 stimulation.
基金supported by the China National Key Hi-Tech Innovation Project for the R&D of Novel Drugs(No.2009ZX09302)National Natural Science Foundation of China(No.81271338)the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20130096110011)
文摘The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines(TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid(p Tα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, q T-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.
文摘Objective： To investigate the changes of cyclin L2（CCNL2） gene mRNA and protein during the differentiation of P19 cells to cardiac myocytes, and to explore the relationship between CCNL2 gene and the differentiation of cardiac myocytes. Methods： P19 cells were cultured with 0.9% DMSO for 18 days. Western blots of cardiac troponin I （cTnI） were used to identify cell differentiation. Total RNA was extracted from P19 cells during the process of differentiation at various time points：pre-differentiation（Day 0）, and Day 1 to Day 18. The expression levels of CCNL2 gene mRNA and protein were evaluated by RT-PCR and Western blot, respectively. Results： After being induced to differentiate by DMSO for 4 days in suspension, spontaneously and rhythmically beating ceils were seen at 8 day, which were cTnI-positive. In P19 cells, both the expression level of CCNL2 gene mRNA and protein were gradually down-regulated. Conclusion： Both the expression of CCNL2 gene and protein were down-regulated during the process of the differentiation of P19 cells into cardiac myocytes, suggesting a possible role for this cyclin in their differentiation.
