The SID-1 double-stranded RNA transporter is not selective for dsRNA length.
The double-stranded RNA (dsRNA) transport protein SID-1 enables systemic RNA interference (RNAi) in Caenorhabditis elegans, whereby silencing initiated by local exposure to dsRNA spreads throughout the animal and to its progeny. Previously, we showed that providing dsRNA in the growth medium of Drosophila S2 cells that express C. elegans SID-1 efficiently triggers RNAi. In these experiments long dsRNA proved to be significantly more effective than short dsRNA in silencing the target gene. Here, we show that equivalent masses of long or short dsRNA accumulate in these cells, indicating that size-dependent silencing is not due to size-selective transport through SID-1. Furthermore, using pulse-chase dsRNA uptake experiments, we show that short dsRNA accumulates more rapidly than long dsRNA. We found that import rates are dependent on dsRNA concentration, consistent with energy-independent, diffusion-limited transport through the SID-1 channel. Comparison of silencing efficiencies between Drosophila S2 cells heterologously expressing SID-1 and primary-cultured C. elegans cells shows similar dsRNA concentration and size dependencies, suggesting that C. elegans regulatory proteins do not measurably enhance or restrict dsRNA transport through SID-1. Finally, we find that coexpressing mutant SID-1 with wild-type SID-1 in S2 cells interferes with SID-1 function, indicating that SID-1 may function as a multimer.
Shih
Joseph D
JD
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Fitzgerald
Michael C
MC
Sutherlin
Marie
M
Hunter
Craig P
CP
D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004331:Drosophila melanogaster; D008565:Membrane Proteins / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; D012330:RNA, Double-Stranded; C452203:SID-1 protein, C elegans