The N-terminal extension of Arabidopsis ARGONAUTE 1 is essential for microRNA activities [mRNA]
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE224451 | GEO | 2023/02/21
REPOSITORIES: GEO
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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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Project description:Argonuate proteins are the central components of the RNA-induced silencing complex (RISC) which execute small RNA-mediated gene regulation mainly in the cytoplasm and their nuclear functions in mammalian cells remain largely unknown. Here we show that in human cancer cells Ago1 is pervasively associated with chromosomal loci throughout the genome with the association centered mostly on actively transcribed promoters. Ago1 but not Ago2 physically interacts with RNA polymerase II (RNAP II) in the nucleus. Moreover, genes bound by Ago1 are significantly enriched for several cancer related pathways. Detailed analysis of these genes including PIK3CA, PRKCH, CDC6 and RRM1 verified Ago1-mediated transcriptional gene regulation through Ago1-promoter interactions. We also found a strong correlation between Ago1-bound genes and genes downregulated by Ago1 depletion which, phenotypically, leads to inhibition of proliferation in different cancer cells. Therefore, our findings reveal the first landscape of human Ago1-chromosomal interactions which may drive oncogenic programs in cancer cells Examination of Ago-DNA interactions in human cancer cells
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