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Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Given the fundamental roles of microRNAs (miRNAs) in physiological, developmental, and pathologic processes, we hypothesized that genes involved in miRNA biogenesis contribute to human complex traits. For 13 such genes, we evaluated the relationship between transcription and 2 classes of complex traits, namely cellular growth and sensitivity to various chemotherapeutic agents in a set of lymphoblastoid cell lines. We found a highly significant correlation between argonaute RNA-induced silencing complex catalytic component 2 (AGO2) expression and cellular growth rate (Bonferroni-adjusted P < 0.05), and report additional miRNA biogenesis genes with suggestive associations with either cellular growth rate or chemotherapeutic sensitivity. AGO2 expression was found to be correlated with multiple drug sensitivity phenotypes. Furthermore, small interfering RNA knockdown of AGO2 resulted in cellular growth inhibition in an ovarian cancer cell line (OVCAR-3), supporting the role of this miRNA biogenesis gene in cell proliferation in cancer cells. Expression quantitative trait loci mapping indicated that genetic variation (in the form of both single-nucleotide polymorphisms and copy number variations) that may regulate the expression of AGO2 can have downstream effects on cellular growth-dependent complex phenotypes.
Copyright © 2015 Elsevier Inc. All rights reserved.
Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.
Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA-protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176-183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 ('slicer' enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson-Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T(M)) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.
Hematopoietic stem cells (HSCs) are characterized by their dual abilities to undergo differentiation into multiple hematopoietic cell lineages or to undergo self-renewal. The molecular basis of these properties remains poorly understood. Recently the piwi gene was found in the embryonic germline stem cells (GSCs) of Drosophila melanogaster and has been shown to be important in GSC self-renewal. This study demonstrated that hiwi, a novel human homologue of piwi, is also present in human CD34(+) hematopoietic progenitor cells but not in more differentiated cell populations. Placing CD34(+) cells into culture conditions that supported differentiation and rapid exit from the stem cell compartment resulted in a loss of hiwi expression by day 5 of a 14-day culture period. Expression of the hiwi gene was detected in many developing fetal and adult tissues. By means of 5' RACE cloning methodology, a novel putative full-length hiwi complementary DNA was cloned from human CD34(+) marrow cells. At the amino acid level, the human HIWI protein was 52% homologous to the Drosophila protein. The transient expression of hiwi in the human leukemia cell line KG1 resulted in a dramatic reduction in cellular proliferation. Overexpression of hiwi led to programmed cell death of KG1 cells as demonstrated by the Annexin V assay system. These studies suggest that hiwi maybe an important negative developmental regulator, which, in part, underlies the unique biologic properties associated with hematopoietic stem and progenitor cells.