RESUMEN
MicroRNAs (miRNAs) are â¼21-nucleotide-long, single-stranded noncoding RNAs that regulate gene expression. Biogenesis of miRNAs is mediated by the two RNase III-like enzymes, Drosha and Dicer. Here we study miRNA biogenesis during maturation of Xenopus oocytes to eggs using microinjection of pri-miRNAs. We show that processing of exogenous and endogenous primary miRNAs (pri-miRNAs) is strongly enhanced upon maturation of oocytes to eggs. Overexpression of cloned Xenopus Drosha in oocytes, however, boosts pri-miRNA processing dramatically, indicating that Drosha is a rate-limiting factor in Xenopus oocytes. This developmental regulation of Drosha is controlled by poly(A) length addition to the Drosha mRNA, which boosts translation upon transition from oocytes to eggs. Processing of pri-miRNAs by Drosha and Dicer has been shown to be affected by adenosine-to-inosine deamination-type RNA editing. Using activated Xenopus eggs for microinjection experiments, we demonstrate that RNA editing can reduce pri-miRNA processing in vivo. This processing block is determined by the structural but not sequence changes introduced by RNA editing.
Asunto(s)
Oocitos/enzimología , Ribonucleasa III/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/enzimología , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , MicroARNs/metabolismo , Datos de Secuencia Molecular , Oocitos/fisiología , Biosíntesis de Proteínas , División del ARN , Edición de ARN , Ribonucleasa III/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. To investigate the effect of dsRNA on silkworm cells, we transfected three kinds of synthetic dsRNAs of 435 bp(Ap1), 300 bp(Ap2) and 399 bp(AH) in length against the various regions of BmNPV's DNA polymerase gene and DNA helicase gene, which are indispensable for viral replication in silkworm cells by TransMessengerTM transfection Reagent. Results indicated that in the experiment where silkworm cells were infected with wild-strain BmNPV of the three dsRNAs, Ap2 and AH can effectively suppress the replication of virus, but Ap1 had no effect on the inhibition of viral replication. Ap2 and AH can reduce the infective titer of BmNPV with a peak change of approximately 3-4 logs on day 4 post-infection. The results of reverse transcript polymerase chain reaction (RT-PCR) and DNA dot blotting also indicated that the expression level of the two target genes and the quantity of viral DNA both distinctly decreased under the influence of Ap2 or AH. Furthermore, using fluorescence microscopy we analyzed the distribution patterns of dsRNA. Our studies revealed that a majority of dsRNA was localized in the nuclear periphery discontinuously after 24 h of transfection.
RESUMEN
Human endostatin is a novel antiangiogenic molecule, which can inhibit the proliferation and development of new blood vessels, and experimentally can cause nearly complete regression of established tumors. In this paper, the cDNA encoding human endostatin was cloned into a baculovirus shuttle vector pBacPAK8 and co-infected with linearized Bm-BacPAK6 DNA into and BmN cells. The recombinant virus was screened and identified by PCR, DNA and RNA dot hybridization, and ELISA assay. The recombinant endostatin was expressed in culture cells, and the larvae and pupa of silkworm by inoculation of recombinant virus. The biological activity assay showed that the expression product in larvae was over 150 microg/ml, about 50-fold higher than that expressed in cultured cells. SDS-PAGE and Western blotting analysis showed a pattern of molecular weight of about 20 kDa. The bio-activity of the protein product was determined by human umbilical vein endothelial cells (ECV304) proliferation test in vitro and the chick chorioallantoic membrane (CAM) vascular inhibition test. Endostatin showed significant inhibitory effect on endothelial cells in a dose-dependent manner. Silkworm-produced endostatin induced apoptosis of endothelial cells and also inhibited angiogenesis in the CAM assay. Combination regimen using angiostatin and endostatin showed more than additive effect in angiogenic inhibition and increasing apoptosis when compared with treatment with the individual antiangiogenic protein.