RNA Interference Effector Proteins Localize to Mobile Cytoplasmic Puncta in Schizosaccharomyces pombe

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Abstract

Ago1, Dcr1 and Rdp1 are the core components of the RNA interference (RNAi) apparatus in the fission yeast Schizosaccharomyces pombe. They function in distinct gene-silencing pathways that direct homology-dependent degradation of mRNA and modification of chromatin. In addition, Ago1 and Dcr1 regulate enactment of Cdc2-dependent cell cycle checkpoints. The ability of the RNAi apparatus to perform multiple roles in these divergent pathways is sure to require dynamic localization of Ago1, Dcr1 and/or Rdp1. Although limited information is available, comprehensive studies regarding the relative localizations of Ago1, Dcr1 and Rdp1 are lacking. To this end, we employed live-cell imaging and immunoelectron microscopy to study the intracellular localizations of these proteins. In contrast to previous reports, our study results indicate that the bulk of Ago1 and Dcr1 form stable complexes and are associated with large, mobile, highly dynamic cytoplasmic elements. The majority of Rdp1 is localized to the nucleus, but a pool of Rdp1 is associated with the same cytoplasmic structures. The movements of these structures were dependent upon ATP and intact microtubules. Recruitment of the RNAi core proteins to these structures was not dependent upon siRNAs. Together, our data indicate that the enzymes required for the initiation and effector phases of RNA-dependent gene silencing are concentrated in a common intracellular location, an arrangement that would be expected to result in highly efficient post-transcriptional gene silencing.

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