Project description:Spodoptera frugiperda is the world’s major agricultural pests, and has the distinctive features of high fecundity, strong migratory capacity and high resistance to most insecticides. At present, the control of S. frugiperda in China relies mainly on the spraying of chemical insecticides. MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNAs, and play crucial regulatory roles in various physiological processes, including the insecticide resistance in insects. However, little is known about the regulatory roles of miRNAs on the resistance of S. frugiperda to insecticides. In the present research, the miRNAs that were differentially expressed after cyantraniliprole, spinetoram, emamectin benzoate and tetraniliprole treatment were analyzed by RNA-Seq. A total of 504 miRNAs were systematically identified from S. frugiperda, and 24, 22, 31 and 30 miRNAs were differentially expressed after treatments of cyantraniliprole, spinetoram, emamectin benzoate and tetraniliprole. GO and KEGG enrichment analyses were used to predict the function of differentially expressed miRNAs’ target genes. Importantly, ten miRNAs were significantly differentially expressed among the treatments of three insecticides. MiR-278-5p, miR-13b-3p, miR-10485-5p and miR-10483-5p were significantly down-regulated among the treatments of three insecticides by RT-qPCR. Furthermore, overexpression of miR-278-5p, miR-13b-3p, miR-10485-5p and miR-10483-5p significantly increased the mortality of S. frugiperda to cyantraniliprole and emamectin benzoate. The mortality was significantly increased with spinetoram treatment after overexpression of miR-13b-3p, miR-10485-5p and miR-10483-5p. These results suggest that miRNAs, which are differentially expressed in response to insecticides, may play a key regulatory role in the insecticide resistance in S. frugiperda.
Project description:Argonaute proteins lie at the heart of the RNA-Induced Silencing Complex (RISC), wherein they use small RNA guides to recognize targets. Some Argonaute proteins can directly cleave their targets, whereas others recruit co-factors to repress independently of “slicing.” Prior studies established the architecture of Argonaute proteins; however, we have not yet had a detailed picture of an Argonaute whose biochemical and biological functions were well established. Here we describe the crystal structure of human Argonaute-2 bound to miR-20a at 2.2 Å resolution. Overall architecture and domain organization is shared with its prokaryotic counterparts, though Ago2 is somewhat more open, with its PAZ domain further removed from the other domains. The miRNA is anchored at both ends by the Mid and PAZ domains but makes several kinks and turns along the binding groove. The RNA confers remarkable stability on the Argonaute protein, locking this otherwise flexible enzyme into a stable conformation.
Project description:Argonaute proteins lie at the heart of the RNA-Induced Silencing Complex (RISC), wherein they use small RNA guides to recognize targets. Some Argonaute proteins can directly cleave their targets, whereas others recruit co-factors to repress independently of “slicing.” Prior studies established the architecture of Argonaute proteins; however, we have not yet had a detailed picture of an Argonaute whose biochemical and biological functions were well established. Here we describe the crystal structure of human Argonaute-2 bound to miR-20a at 2.2 Å resolution. Overall architecture and domain organization is shared with its prokaryotic counterparts, though Ago2 is somewhat more open, with its PAZ domain further removed from the other domains. The miRNA is anchored at both ends by the Mid and PAZ domains but makes several kinks and turns along the binding groove. The RNA confers remarkable stability on the Argonaute protein, locking this otherwise flexible enzyme into a stable conformation. total small RNAs (19-29nt) and small RNAs associated with purified hArgonaute2 purified from SF9 cells. These were processed and sequenced on Illumina GA-II platform.
Project description:Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. In most eukaryotes, kinetochore assembly is primed by the histone H3 variant CenH3, which physically interacts with components of the inner kinetochore constitutive-centromere-associated-network (CCAN). Unexpected to its critical function, previous work identified that select eukaryotic lineages, including several insects, have lost CenH3, while having retained homologs of the CCAN. These findings imply alternative CCAN assembly pathways in these organisms that function in CenH3-independent manners. Here, we study the composition and assembly of CenH3-independent kinetochores of Lepidoptera. We show that lepidopteran kinetochores consist of previously identified CCAN homologs as well as additional components including a divergent CENP-T homolog, which are required for accurate mitotic progression. Our study focuses on CENP-T that we find both necessary and sufficient to recruit the Mis12 outer kinetochore complex. In addition, CRISPR-mediated gene editing in Bombyx mori establishes an essential function of CENP-T in vivo. Finally, the retention of CENP-T homologs in other independently-derived CenH3-deficient insects indicates a conserved mechanism of kinetochore assembly between these lineages. Our study provides the first functional insights into CCAN-based kinetochore assembly pathways that function independently of CenH3, thus contributing to the emerging picture of an unexpected plasticity to build a kinetochore.
Project description:Several members from microRNA 17-92 cluster, i.e. miR-19a, miR-19b and miR-20a, were found up-regulated in human epidermal keratinocytes at wound-edges compared to the intact skin; however their biological role in keratinocytes during wound repair has not been studied. To study the genes regulated by miR-19a, miR-19b and miR-20a, we transfected miRNA specific mimics, i.e. pre-miR-19a, pre-miR-19b or pre-miR-20a into human primary epidermal keratinocytes to overexpress them. We performed a global transcriptome analysis of keratinocytes upon overexpression of miR-19a or miR-19b or miR-20a using Affymetrix arrays.
Project description:Identify potential miR-20a regulated mRNAs and linked pathways in the setting of QK knockdown by comparing the transcriptional profiles of shQK-transduced human U87 cells together with miR-20a or a scrambled miRNA control (miR-NT)
Project description:Identify potential miR-20a regulated mRNAs and linked pathways in the setting of QK knockdown by comparing the transcriptional profiles of shQK-transduced human Hs683 cells together with miR-20a or a scrambled miRNA control (miR-NT)
Project description:In this study, we evaluated genetic mouse models of inducible renal epithelia-specific miR-17~92 loss-of-function and gain-of-function using unilateral ureteral obstruction. We utilized PAR-CLIP in HK2 cells to identify miR-17/-20a targets, which we subsequently validated in vitro. We examined expression of a novel miR-17/-20a target in human nephrectomy samples with varying degrees of fibrosis. We identify an activator of Hippo signaling, FERM domain-containing protein 6 (FRMD6, also known as Willin), as a novel miR-17/20a target. Frmd6 is upregulated in tubular epithelium of obstructed kidneys that lack miR-17~92, along with increased phosphorylation of Smad3 and STAT3, two known miR-17~92 profibrotic targets. Frmd6 overexpression is sufficient to result in elevated secretion of the extracellular matrix component collagen III in vitro. Finally, we demonstrate that FRMD6 expression is associated with collagen III expression in human nephrectomy samples.Our findings demonstrate that the miR-17~92 cluster in renal epithelia functions in an anti-fibrotic manner by regulating multiple pro-fibrotic pathways. We also identify Frmd6 as a novel miR-17/20a target in renal epithelia, which may drive renal fibrosis.