Systemic RNA Interference Deficiency-1 (SID-1) extracellular domain selectively binds long double-stranded RNA and is required for RNA transport by SID-1 [Molecular Biophysics]

June 11th, 2015 by Li, W., Koutmou, K. S., Leahy, D. J., Li, M.

During systemic RNA interference (RNAi) in C. elegans, RNA spreads across different cells and tissues in a process that requires systemic RNA interference deficient-1 (sid-1) gene that encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 (S2) cells enables passive uptake of double-stranded RNA (dsRNA) and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as a RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1 mediated RNA transport efficiency, providing evidence for that dsRNA binding to SID-1 ECD is related to RNA transport. To examine functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive for dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.
  • Posted in Journal of Biological Chemistry, Publications
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