(L to R) Craig Hunter, Deborah McEwan, and Alexandra Weisman
What if your lunch could tell your body to shut off a particular gene? This is exactly what happens for the microscopic worm Caenorhabditis elegans. When these worms eat food that contains double-stranded RNAs (dsRNA), their cells stop expressing any genes that contain the same sequence as that dsRNA through a process termed environmental RNA interference (RNAi). Almost immediately after its discovery, environmental RNAi became a widespread research technique and quickly revolutionized genetic screening in both C. elegans and many other model organisms. More recently, environmental RNAi strategies have been applied to manage large invertebrate populations, acting as either a novel pest-specific pesticide or to prevent or treat viral outbreaks such as those that have been linked to colony collapse in honeybees. While the potency and specificity of environmental RNAi continues to hold great promise for gene-specific drugs and designer pesticides, intracellular delivery remains a challenge for most animals including humans (the delivery problem). The Hunter lab studies this problem by learning how C. elegans imports and distributes environmental dsRNAs to cells throughout their body. Coupling therapeutic RNAi delivery to endogenous RNA transport pathways may help to someday overcome this delivery problem.
A previous genetic screen in the Hunter lab discovered proteins required for environmental RNAi in C. elegans. The best characterized of these is SID-1, a dsRNA channel that must be present for dsRNA to enter into any cell. In a Molecular Cell article published online this month (McEwan et al Mol Cell 2012), members of Craig Hunter’s lab have determined that a second protein, SID-2, is also a dsRNA transporter. Unlike the broadly-expressed SID-1 protein, SID-2 is only present in intestinal cells and is only required to import ingested dsRNAs from the intestinal lumen. This discovery, however, led to a mystery: since either RNA transporter is sufficient to internalize dsRNA, why are both SID-1 and SID-2 required for environmental RNAi? Using genetic and biochemical approaches, Hunter and colleagues found that these two dsRNA transporters mostly likely act at mechanistically distinct steps; SID-2 acts first to selectively endocytose ingested dsRNA into transport vesicles and SID-1 functions later to bring the dsRNA into the cytoplasm, either transporting dsRNA directly from the vesicles or by importing dsRNA from the body cavity space following the exocytosis of the vesicularized dsRNA. SID-1 is a widely conserved protein and mouse and human SID-1 homologs have been implicated in dsRNA transport. In contrast SID-2 homologs are present only in Caenorhabditis nematodes. Interestingly, studies in mice show that coupling dsRNA to ligands that bind to endogenous endocytosis receptors enhances dsRNA uptake and RNAi silencing. Together, these observations suggest that the widely observed phenomena of environmental RNAi may rely on a multi-step process that begins with a SID-2-like step that endocytoses dsRNA into vesicles followed by dsRNA transport via SID-1 that releases the dsRNA into the cytoplasm to trigger gene silencing.
Also published by the Hunter lab this month are two papers that report the identification of SID-3 (a cdc42 activated kinase Jose et al PNAS 2012) and SID-5 (a late endosomal protein [Hinas et al Curr Biol 2012 (in press)]). Unlike sid-1 and sid-2, environmental RNAi is only partially disrupted in sid-3 and sid-5 mutants, indicating that these proteins may regulate the activity and localization of the SID-1 and SID-2 RNA transporters. Further characterization of the regulation and function of extracellular RNA transport pathways will provide additional insight into the development of therapeutic RNAi.
Read more in Molecular Cell