Project description:Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.
Project description:SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal secA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.
Project description:Argonaute/Piwi proteins associate with small RNAs that typically provide sequence specificity for RNP function in gene and genome regulation. Here we show that Twi12, a Tetrahymena Piwi protein essential for growth, is loaded with mature tRNA fragments. The tightly bound ~18-22 nt tRNA 3M-bM-^@M-^Y fragments are biochemically distinct from the tRNA halves produced transiently in response to stress. Notably, the end positions of Twi12-bound tRNA 3' fragments precisely match RNAs detected in total small RNA of mouse embryonic stem cells and human cancer cells. Our studies demonstrate unanticipated evolutionary conservation of mature tRNA processing to tRNA-fragment small RNAs. Two libraries are analyzed here: sRNAs associated with slightly overexpressed ZZ-tagged Twi12 purified under native conditions (size range 15-34nt), and those associated after formaldehyde crosslinking (15-22nt).
Project description:In order to determine the impact of L3mbtl2 on genome wide mRNA expression microarray analysis was carried out in L3mbtl2 knock out ES cells and wild type ES cells. The same analysis was also carried out in knock out cells infected with L3mbtl2-F vector and a control vector. Experiment to study targets of L3mbtl2 in C57bl6/129 hybrid ES cells. 4 samples: wild type, L3mbtl2 knock out, L3mbtl2 knock out rescue and rescue with empty vector
Project description:Argonaute2 proteins (Ago2) are key component of RNA-induced gene silencing complexes (RISCs), which is crucial for microRNAs to repress target genes. The function of Ago2 for oogenesis is unknown due to early embryonic lethal of Ago2 knockout mice. Here we show the effect of loss of Ago2 in mouse oogenesis by specific deletion of it in growing oocyte. Although the Ago2-deficient oocytes can develop to mature oocytes, they have abnormal spindle and the chromosomes that can’t cluster together properly, which are very similar to the phenotype of Dicer-deficient oocytes. We checked miRNA expression profile and found that in Ago2-deficient oocyte, the expression of most miRNAs reduced more than 80%. To understand the downstream genes regulated by Ago2, we used microarray on Ago2-deficient oocyte and found that 512 genes were upregulated and 1073 genes were downregulated (FC>2, P<0.05). Our data proved that Ago2 have a key function for mouse oogenesis through globally regulating miRNA stability and function.
Project description:Argonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes.Ago2 was deleted specifically in the growing oocytes. Although the Ago2-deficient oocytes are able to develop to mature oocytes, they have abnormal spindles and chromosomes that are unable to cluster together properly. This phenotype is very similar to the phenotype of Dicer-deficient oocytes. We examined the microRNA expression profile in the Ago2-deficient oocyte and found that the expression of most microRNAs was reduced by more than 80%. To determine the downstream genes that are regulated by Ago2, we used microarray analysis on Ago2-deficient oocytes and found that 512 genes were upregulated and 1,073 genes were downregulated (FC > 2, P < 0.05).Our study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes.
Project description:Essential genes, those critical for the survival of an organism under certain conditions, play a significant role in pharmaceutics and synthetic biology. Knowledge of protein localization is invaluable for understanding their function as well as the interaction of different proteins. However, systematical examination of essential genes from the aspect of the localizations of proteins they encode has not been explored before. Here, a comprehensive protein localization analysis of essential genes in 27 prokaryotes including 24 bacteria, 2 mycoplasmas and 1 archaeon has been performed. Both statistical analysis of localization information in these genomes and GO (Gene Ontology) terms enriched in the essential genes show that proteins encoded by essential genes are enriched in internal location sites, while exist in cell envelope with a lower proportion compared with non-essential ones. Meanwhile, there are few essential proteins in the external subcellular location sites such as flagellum and fimbrium, and proteins encoded by non-essential genes tend to have diverse localizations. These results would provide further insights into the understanding of fundamental functions needed to support a cellular life and improve gene essentiality prediction by taking the protein localization and enriched GO terms into consideration.
Project description:Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGF?, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.
Project description:Dmbx1 is a paired-class homeodomain transcription factor. We show here that mice deficient in Dmbx1 exhibit severe leanness associated with hypophagia and hyperactivity and that isolation of a Dmbx1(-/-) mouse from its cohabitants induces self-starvation, sometimes leading to death, features similar to those of anorexia nervosa in humans. Interestingly, overexpression of agouti in Dmbx1(-/-) mice failed to induce aspects of the A(y)/a phenotype, including hyperphagia, obesity, and diabetes mellitus. In Dmbx1(-/-) mice, administration of agouti-related protein increased cumulative food intake for the initial 6 h but significantly decreased it over 24- and 48-h periods. In addition, Dmbx1 was shown to be expressed at embryonic day 15.5 in the lateral parabrachial nucleus, the rostral nucleus of the tractus solitarius, the dorsal motor nucleus of the vagus, and the reticular nucleus in the brainstem, all of which receive melanocortin signaling, indicating involvement of Dmbx1 in the development of the neural network for the signaling. Thus, Dmbx1 is essential for various actions of agouti-related protein and plays a role in normal regulation of energy homeostasis and behavior.