04-083 Sigma-AldrichAnti-Ago1 Antibody, clone 6D8.2

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.Cell Research advance online publication 25 March 2014; doi:10.1038/cr.2014.36.

The roles of Argonaute proteins in cytoplasmic microRNA and RNAi pathways are well established. However, their implication in small RNA-mediated transcriptional gene silencing in the mammalian cell nucleus is less understood. We have recently shown that intronic siRNAs cause chromatin modifications that inhibit RNA polymerase II elongation and modulate alternative splicing in an Argonaute-1 (AGO1)-dependent manner. Here we used chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) to investigate the genome-wide distribution of AGO1 nuclear targets. Unexpectedly, we found that about 80% of AGO1 clusters are associated with cell-type-specific transcriptional enhancers, most of them (73%) overlapping active enhancers. This association seems to be mediated by long, rather than short, enhancer RNAs and to be more prominent in intragenic, rather than intergenic, enhancers. Paradoxically, crossing ChIP-seq with RNA-seq data upon AGO1 depletion revealed that enhancer-bound AGO1 is not linked to the global regulation of gene transcription but to the control of constitutive and alternative splicing, which was confirmed by an individual gene analysis explaining how AGO1 controls inclusion levels of the cassette exon 107 in the SYNE2 gene.

Long noncoding RNAs (lncRNAs) are known to regulate neighboring protein-coding genes by directing chromatin remodeling complexes, imprinting, and X-chromosome inactivation. In this study, we explore the function of lncRNAs in small RNA-triggered transcriptional gene activation (TGA), a process in which microRNAs (miRNAs) or small interfering RNAs (siRNAs) associated with Argonaute (Ago) proteins induce chromatin remodeling and gene activation at promoters with sequence complementarity. We designed a model system with different lncRNA and chromatin environments to elucidate the molecular mechanisms required for mammalian TGA. Using RNA-fluorescence in situ hybridization (FISH) and rapid amplification of cDNA ends (RACE)-PCR, we demonstrated that small RNA-triggered TGA occurs at sites where antisense lncRNAs are transcribed through the reporter gene and promoter. Small RNA-induced TGA coincided with the enrichment of Ago2 at the promoter region, but Ago2-mediated cleavage of antisense lncRNAs was not observed. Moreover, we examined the allele-specific effects of lncRNAs through a Cre-induced inversion of a poly(A) sequence that was designed to block the transcription of antisense lncRNAs through the reporter gene region in an inducible and reversible manner. Termination of nascent antisense lncRNAs abrogated gene activation triggered by small RNAs, and only allele-specific cis-acting antisense lncRNAs, but not trans-acting lncRNAs, were capable of rescuing TGA. Hence, this model revealed that antisense lncRNAs can mediate TGA in cis and not in trans, serving as a molecular scaffold for a small RNA-Ago2 complex and chromatin remodeling.

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.

Drosha-processed microRNAs (miRNAs) have been shown to be exported from the nucleus to the cytoplasm by Exportin 5, where they are processed a second time to generate mature miRNAs. In this work we show that miRNAs also use CRM1 for nuclear-cytoplasmic shuttling. Inhibition of CRM1 by Leptomycin B results in nuclear accumulation of miRNA guide sequences. Nuclear to cytoplasmic transport can be actively competed by synthetic small interfering RNAs, indicating that this pathway is shared by different classes of processed small RNAs. We also find that CRM1 coimmunoprecipitates with Ago-1, Ago-2, Topo2alpha, EzH2, and Mta, consistent with a role of Argonautes and small RNAs in chromatin remodeling.

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe(3)O(4)-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes.