Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling in cells with Ago1 knockdown (Affymetrix)

ABSTRACT: Argonautes, a family of highly evolutionarily conserved proteins, are the central platform for small RNA-mediated gene regulatory mechanisms which occur mainly in the cytoplasm. To understand a potential role of Argonaute 1 (Ago1) protein in the nucleus of mammalian cells in regulating gene transcription and epigenetics, we performed integrated analyses of Ago1 ChIP-sequencing (GSE40536) and gene expression profiling in cells depleted of Ago1. For gene expression profiling, we knocked down the expression of Ago1 by siRNA in PC-3 cells and compared gene expression profiles in the cells depleted of Ago1 and cells receiving control treatments. We found that Ago1 depletion resulted in more downregulated genes which were enriched in gene responsible for promoting cell cycle progression, DNA replication and repair, and response to endogenous stimuli. Integrated analyses of Ago1-chromosomal interactions and gene expression changes in response to Ago1 depletion reveal a significant correlation between Ago1-bound genes and genes altered by Ago1 depletion. These genes are significantly enriched in cancer-related pathways. Our data suggests a nuclear function of Ago1 in regulating gene transcription. PC-3 cells were mock transfected or transfected with an Ago1 specific siRNA or a control siRNA. Each treatment was done in duplicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Vera Huang

PROVIDER: E-GEOD-42600 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis or kala-azar, the most severe form of leishmaniasis in humans. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA interference—collectively RNA silencing pathways—participates in the regulation of various biological processes in most eukaryotic cells. Complexes of Argonaute proteins with small RNAs are core components of the RNA interference system and play a key role in silencing gene expression. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference pathways to promote their survival. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Western blot analysis showed that protein abundance of infected macrophage Argonaute 1 (Ago1) was selectively and significantly higher than that of non-infected control at 24 h post-infection, suggesting that Ago1 plays a role in pathogenicity. In fact, siRNA-mediated downregulation of Ago1 enhanced Leishmania clearance from infected host cells, linking macrophage Ago1 to Leishmania virulence. To investigate the mechanisms of host Ago1 in Leishmania pathogenesis, a stable isotope labeling by amino acids in cell culture (SILAC)-based whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, the proteomic-based detailed biochemical analysis showed that Leishmania modulated host RNA-induced silencing complex (RISC) composition during infection, strongly suggesting macrophage RISC targeting. Strikingly, Leishmania proteins were detected as part of host RISC in infected cells. Together, our results demonstrate that Leishmania targets host RNA interference machinery to promote its survival inside the host macrophage.

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Gene expression profiling in cells with Ago1 knockdown (Affymetrix)

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