| In C. elegans, double-stranded RNA (dsRNA) introduced by injection, transgene expression, or from the environment can cross cellular membranes and trigger sequence-specific gene silencing by RNA interference (RNAi). Although this intercellular transport is already being used for many applications, it is poorly understood. In C. elegans, at least two membrane proteins are required to import environmental dsRNA, the broadly-expressed dsRNA channel SID-1 and the apically-localized intestinal protein SID-2. To further characterize SID-2 and to understand how SID-1 functions with a tissue-specific protein, I expressed SID-2 in Drosophila S2 cells and tested whether it could transport dsRNA independent of SID-1. In these cells, an acidic environment similar to the lumenal space enables SID-2 to selectively internalize long dsRNA by an endocytic process. Furthermore, structure-functional analysis of SID-2 in S2 cells and in C. elegans demonstrates that extracellular histidine residues are necessary for this transport, potentially by interacting with the dsRNA phosphate backbone. Since SID-2 functions independently of SID-1, these proteins likely act sequentially during dsRNA internalization with SID-2 importing dsRNA directly from the lumenal space. The discovery that SID-1 and SID-2 interact in yeast suggests their activities may be functionally coupled. This may represent a conserved mechanism in which dsRNA is imported by an endocytic process linked to SID-1 transport. To determine whether there are other C. elegans dsRNA endocytic receptors and to find additional RNAi transport components, I performed a yeast two-hybrid screen and identified 75 proteins not previously associated with RNAi transport that interact with SID-1. Further analysis of these interacting proteins is also described. |
