Abstract
Growth and proliferation of tumor cells requires coordinated activation of many cellular processes, including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation to ...set thresholds for expression of target proteins. Emerging data suggest that microRNA function, like cap-dependent translation, is enhanced by activation of cancer-associated signaling due, at least in part, to induction of GW182 protein expression. In the current study, we demonstrate that increased expression of GW182 in normal and transformed hematopoietic cells contributes to enhanced microRNA function by increasing overall capacity for microRNA-mediated target repression. Furthermore, expression of GW182 was found to be regulated post-transcriptionally by the translation initiation complex downstream of convergent signaling through the PI3K/Akt/mTOR and Jak/Stat/Pim signaling pathways, both of which are commonly activated in hematopoietic malignancies. PI3K/Akt/mTOR signaling contributed to GW182 expression primarily by stimulating eIF4E binding to GW182 mRNA, while Jak/Stat/Pim signaling enhanced GW182 expression by additionally increasing eIF4A-dependent unwinding of G-quadraplexes in the 5′ untranslated region (UTR) of GW182 mRNA. Inhibition of either eIF4E-dependent translation initiation by treatment with rapamycin or eIF4A-dependent unwinding of the GW182 5′ UTR by treatment with silvestrol led to decreased GW182 expression and microRNA function. Our data provide a mechanism by which microRNA function is linked to cap-dependent translation, allowing thresholds for expression of microRNA targeted proteins to be maintained despite changes in protein synthesis.
Citation Format: Scott H. Olejniczak, Gaspare La Rocca, Megan Radler, Craig B. Thompson. Coordinated regulation of cap-dependent translation and microRNA function by convergent signaling through the PI3K/Akt/mTOR and Jak/Stat/Pim signaling pathways. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 973. doi:10.1158/1538-7445.AM2015-973
The identification of miRNA targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these ...methods are technically challenging and not easily applicable to an
in vivo
setting.
To overcome these limitations and facilitate the investigation of miRNA functions
in vivo
, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers.
This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks
in vivo
under physiological and pathological conditions.
Li, Pritykin, Concepcion et al. report the development of Halo-enhanced Ago2 pulldown (HEAP), a method that streamlines the experimental identification of Ago2-miRNA-mRNA interaction sites in murine cells and tissues.
Abstract
Pediatric posterior fossa ependymomas are poorly understood childhood brain tumors and have no effective treatments. The biology of these tumors is obscure as recent sequencing efforts ...suggest that they lack recurrent genetic alterations. A subset of these tumors termed PF-A ependymomas exhibits CpG-island hypermethylation implicating epigenetic alterations in their pathogenesis. Through comprehensive analyses of histone modification, we discovered global H3K27me3 reduction in a subset of these tumors. Tumors with lowered H3K27me3 showed many clinical and biologic similarities with PFA-ependymomas. Global reduction in H3K27me3 is likewise observed in pediatric gliomas that bear histone H3K27M mutations termed diffuse intrinsic pontine gliomas (DIPG) that also arise in the posterior fossa of young children. Analyses of ependymomas with reduced H3K27me3 and H3K27M mutant DIPGs showed many similarities in DNA methylation and enrichment of H3K27me3 in many genomic loci important for neuroglial specification. Combined integrative analysis of both tumor types uncovered common epigenetic deregulation of select factors that control radial glial biology and radial glia in the developing posterior fossa showed reduced H3K27me3. Finally, PF ependymomas with lowered H3K27me3 were more invasive radiologically and exhibited poor prognosis in three independent cohorts (P<0.001 in all three cohorts, collective n>300). These data have clinical implications for biomarker development and to inform epigenetic approaches to treat PF ependymomas.
Citation Format: Sriram Venneti, Jill Bayliss, Piali Mukherjee, Chao Lu, Siddhant Jain, Chan Chung, Daniel Martinez, Benjamin Sabari, Ashley Margol, Pooja Panwalkar, Abhijit Paroloia, Melike Pekmezci, Richard Mc Eachin, Marcin Cieslik, Benita Tamrazi, Benjamin Garcia, Gaspare La Rocca, Mariarita Santi, Peter Lewis, Cynthia Hawkins, Ari Melnick, C David Allis, Craig B. Thompson, Arul Chinnaiyan, Alexander R. Judkins. A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2017-3863
Preparation of protein nuclear extracts is often the first step to study in vitro biological processes occurring in the nucleus of the eukaryotic cell. Nuclear extracts have been extensively used in ...different model organisms to identify and study protein function in nuclei. Drosophila embryos can be collected in large quantities and have been the source of choice for the production of protein nuclear extracts. However, most of Drosophila in vivo studies on protein function are conducted in larval tissues. Here we report a new method to produce highly stable large-scale protein nuclear extracts from whole Drosophila larvae that are suited for a variety of biochemical analyses.
MicroRNA145 (miR145), a tumor suppressor miR, has been reported to inhibit growth of human cancer cells, to induce differentiation and to cause apoptosis, all conditions that result in growth arrest. ...In order to clarify the functional effects of miR145, we have investigated its expression in diverse conditions and different cell lines. Our results show that miR145 levels definitely increase in differentiating cells and also in growth-arrested cells, even in the absence of differentiation. Increased expression during differentiation sometimes occurs as a late event, suggesting that miR145 could be required either early or late during the differentiation process.
Abstract
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the world. The TCL1 gene, responsible for prolymphocytic T cell leukemia, is also overexpressed in human B cell malignancies ...and overexpression of the Tcl1 protein occurs frequently in CLL. Aging transgenic mice that overexpress TCL1 under control of the μ immunoglobulin gene enhancer, develop a CD5+ B cell lymphoproliferative disorder mimicking human CLL and implicating TCL1 in the pathogenesis of CLL. In the current study, we exploited this transgenic mouse to investigate two different CLL-related issues: potential treatment of CLL and characterization of neoplasms that accompany CLL. We successfully transplanted CLL cells into syngeneic mice that led to CLL development in the recipient mice. This approach allowed us to verify the involvement of the Tcl1/Akt/mTOR biochemical pathway in the disease by testing the ability of a specific pharmacologic agent, rapamycin, to slow CLL. We also showed that 36% of these transgenic mice were affected by solid malignancies, in which the expression of the Tcl1 protein was absent. These findings indicate that other oncogenic mechanism(s) may be involved in the development of solid tumors in Eμ-TCL1 transgenic mice. (Cancer Res 2006: 66(2): 915–20)
MicroRNAs (miRs) modulate gene expression by repressing translation or inducing degradation of targeted mRNAs. Although miRs are currently explored as novel therapeutic targets, the cell type and ...context-specific effects of miRs in the heart are not well explored. Therefore, we determined target regulation using miR-92a as an example. Inhibition of miR-92a was shown to augment neovascularization and recovery after acute myocardial infarction (MI).We performed microarrays using RNA isolated from the apex of control mice (n=4), healthy mice treated with the miR-92a-inhibitior LNA-92a (2.5 mg/kg) (n=4), and mice at 4 days after MI treated with either PBS (n=4) or LNA-92a (n=3). miR-92a was inhibited by more than 95 % in all LNA-92a-treated mice. In LNA-92a treated control mice, 73 genes including only one predicted miR-92a target were significantly regulated. In mice after MI, 310 genes (fold change<+/-1.4;p<0.05) were regulated by LNA-92a including 36 predicted miR-92a targets such as cardioprotective Hmox1 and the transcription factor Id2, which is involved in organ regeneration. A subset of 10 candidates was validated by qRT-PCR. Consistent with a more profound regulation of miR-92a targets after MI, miR-92a was enriched by 2-fold in the risk complex in the infarct tissue compared to remote or healthy tissue. Since miRs might exhibit distinct effects in specific cell types, we additionally isolated cell populations from mouse hearts after MI and determined gene expression by RNA sequencing. LNA-92a treatment upregulates 64 predicted miR-92a targets in cardiomyocytes, 68 in endothelial cells, 63 in fibroblasts, whereas only 17 predicted targets were regulated in immune cells. Most targets were regulated in a cell type specific manner and none was regulated in all cell types.In conclusion, miR-92a inhibition has context and cell type specific effects in the heart. The more profound regulation of miR-92a targets and enrichment of miR-92a in the risk complex in infarcted tissue suggests that tissue injury amplifies the response to miR-92a inhibition. The cell type specific regulation of miR-92a targets implies that deciphering the specific functions of a given miR in cell populations is essential to understand the overall impact of miRs in complex tissues.
Nucleosome remodeling and covalent modifications of histones play fundamental roles in chromatin structure and function. However, much remains to be learned about how the action of ATP-dependent ...chromatin remodeling factors and histone-modifying enzymes is coordinated to modulate chromatin organization and transcription. The evolutionarily conserved ATP-dependent chromatin-remodeling factor ISWI plays essential roles in chromosome organization, DNA replication, and transcription regulation. To gain insight into regulation and mechanism of action of ISWI, we conducted an unbiased genetic screen to identify factors with which it interacts in vivo. We found that ISWI interacts with a network of factors that escaped detection in previous biochemical analyses, including the Sin3A gene. The Sin3A protein and the histone deacetylase Rpd3 are part of a conserved histone deacetylase complex involved in transcriptional repression. ISWI and the Sin3A/Rpd3 complex co-localize at specific chromosome domains. Loss of ISWI activity causes a reduction in the binding of the Sin3A/Rpd3 complex to chromatin. Biochemical analysis showed that the ISWI physically interacts with the histone deacetylase activity of the Sin3A/Rpd3 complex. Consistent with these findings, the acetylation of histone H4 is altered when ISWI activity is perturbed in vivo. These findings suggest that ISWI associates with the Sin3A/Rpd3 complex to support its function in vivo.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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