Argonautes (AGOs) are a highly conserved family of proteins found in most eukaryotes and involved in mechanisms of gene regulation, both at the transcriptional and post-transcriptional level. Among ...other functions, AGO proteins associate with microRNAs (miRNAs) to mediate the post-transcriptional repression of protein-coding genes. In this process, AGOs associate with members of the trinucleotide repeat containing 6 protein (TNRC6) family to form the core of the RNA-induced silencing complex (RISC), the effector machinery that mediates miRNA function. However, the description of the exact composition of the RISC has been a challenging task due to the fact the AGO's interactome is dynamically regulated in a cell type- and condition-specific manner. Here, we summarize some of the most significant studies that have identified AGO complexes in mammalian cells, as well as the approaches used to characterize them. Finally, we discuss possible opportunities to exploit what we have learned on the properties of the RISC to develop novel anti-cancer therapies. SIGNIFICANCE STATEMENT: The RNA-induced silencing complex (RISC) is the molecular machinery that mediates miRNA function in mammals. Studies over the past two decades have shed light on important biochemical and functional properties of this complex. However, many aspects of this complex await further elucidation, mostly due to technical limitations that have hindered full characterization. Here, we summarize some of the most significant studies on the mammalian RISC and discuss possible sources of biases in the approaches used to characterize it.
Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain ...and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.
Ars2 is a component of the nuclear cap-binding complex that contributes to microRNA biogenesis and is required for cellular proliferation. Here, we expand on the repertoire of Ars2-dependent ...microRNAs and determine that Ars2 regulates a number of mRNAs, the largest defined subset of which code for histones. Histone mRNAs are unique among mammalian mRNAs because they are not normally polyadenylated but, rather, are cleaved following a 3′ stem loop. A significant reduction in correctly processed histone mRNAs was observed following Ars2 depletion, concurrent with an increase in polyadenylated histone transcripts. Furthermore, Ars2 physically associated with histone mRNAs and the noncoding RNA 7SK. Knockdown of 7SK led to an enhanced ratio of cleaved to polyadenylated histone transcripts, an effect dependent on Ars2. Together, the data demonstrate that Ars2 contributes to histone mRNA 3′ end formation and expression and these functional properties of Ars2 are negatively regulated by interaction with 7SK RNA.
► Ars2 binds histone mRNAs and facilitates their 3′ end processing and expression ► 7SK RNA binds Ars2 and negatively affects histone mRNA 3′ end processing ► In addition to histone mRNAs, Ars2 regulates a subset of microRNAs
K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic ...K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.
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•Oncogenic K-Ras induces global de-repression of physiological miRNA targets in vivo•K-RasG12D enhances Ago2:mRNA binding, likely through Ago2 hypo-phosphorylation•K-RasG12D transcriptionally suppresses Csnk1a1 and Csnk2a1 expression/activities•Csnk1a1 and Csnk2a1 may hierarchically cooperate in their phosphorylation of Ago2
Shui et al. report the in vivo de-repression of global miRNA targets induced by the oncogenic K-Ras (K-RasG12D) that is paradoxically accompanied by the enhanced Ago2:mRNA binding. Integrating in vivo modeling with omics network analysis, they mechanistically connect the K-Ras oncogene to the dysregulation of hundreds of downstream RNA/proteins.
The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates ...microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have shed light on the role of AGO and TNRC6 in mammalian chromatin biology and on the implications these novel mechanisms may have in our understanding of regulating gene expression.
Glucose and glutamine are the two principal nutrients that cancer cells use to proliferate and survive. Many cancers show altered glucose metabolism, which constitutes the basis for in vivo positron ...emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)F-FDG). However, (18)F-FDG is ineffective in evaluating gliomas because of high background uptake in the brain. Glutamine metabolism is also altered in many cancers, and we demonstrate that PET imaging in vivo with the glutamine analog 4-(18)F-(2S,4R)-fluoroglutamine ((18)F-FGln) shows high uptake in gliomas but low background brain uptake, facilitating clear tumor delineation. Chemo/radiation therapy reduced (18)F-FGln tumor avidity, corresponding with decreased tumor burden. (18)F-FGln uptake was not observed in animals with a permeable blood-brain barrier or neuroinflammation. We translated these findings to human subjects, where (18)F-FGln showed high tumor/background ratios with minimal uptake in the surrounding brain in human glioma patients with progressive disease. These data suggest that (18)F-FGln is avidly taken up by gliomas, can be used to assess metabolic nutrient uptake in gliomas in vivo, and may serve as a valuable tool in the clinical management of gliomas.
Cell growth and proliferation require the coordinated activation of many cellular processes, including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation and set thresholds ...for expression of target proteins. Emerging data suggest that microRNA function is enhanced by cellular activation due in part to induction of the RNA-induced silencing complex (RISC) scaffold protein GW182. In the current study, we demonstrate that increased expression of GW182 in activated or transformed immune cells results from effects of phosphoinositol 3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) and Jak-Stat-Pim signaling on the translation of GW182 mRNA. Both signaling pathways enhanced polysome occupancy and eukaryotic initiation factor 4E (eIF4E) binding to the 5′ 7mG cap of GW182 mRNA. The effect of Jak-Stat-Pim signaling on polysome occupancy and expression of GW182 protein was greater than that of PI3K-Akt-mTOR signaling, likely resulting from enhanced eIF4A-dependent unwinding of G-quadruplexes in the 5′ untranslated region of GW182 mRNA. Consistent with this, GW182 expression and microRNA function were reduced by inhibition of mTOR or Pim kinases, translation initiation complex assembly, or eIF4A function. Taken together, these data provide a mechanistic link between microRNA function and cap-dependent translation that allows activated immune cells to maintain microRNA-mediated repression of targets despite enhanced rates of protein synthesis.
Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) ...hypermethylation PF group A (PFA), implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.
ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is ...largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, ...Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.
Significance MicroRNAs limit gene expression by recruiting a large protein complex known as the RNA-induced silencing complex (RISC) to target mRNAs. While attempting to understand physiological regulation of RISC assembly, we found that most healthy adult tissues retain a reserve of microRNAs not stably associated with target mRNA. Recruitment of microRNAs to large mRNA-containing complexes was accompanied by an increase in their ability to repress targets and was regulated in part by phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway-dependent enhancement of the glycine-tryptophan protein of 182 kDa protein expression. Data presented here suggest that in vivo, many expressed microRNAs exist in an inactive reserve, allowing resting cells to use microRNAs to dynamically regulate the translation of target mRNAs in their environment.
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