ATP-binding Cassette Transporters Are Required for Efficient RNA Interference inCaenorhabditis elegans

Abstract

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC) transporter gene superfamily. ABC transporters use ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells, organellar biogenesis, and mechanisms that protect against viral infection. HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that eight additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that use ABC proteins.

Role of the Caenorhabditis elegans Multidrug Resistance Gene, mrp-4, in Gut Granule Differentiation

“…However, it is clear that MRP transporters also mediate the movement of a variety of materials across cellular membranes including glutathione conjugates, bile salts, and nucleotides (Borst and Elferink 2002;Deeley et al 2006;Kruh and Belinsky 2003). While the transport specificity of C. elegans MRPs are unclear, they are known to be necessary for heavy metal resistance (Broeks et al 1996), dauer formation (Yabe et al 2005), and RNAi (Sundaram et al 2006a). C. elegans mrp-4 has recently been shown to contribute to the cellular defects and embryonic lethality resulting from loss of cup-5 function (Schaheen et al 2006).…”

“…Functions for 18 C. elegans ABC transporters have been defined through genetic studies. Like humans, C. elegans ABC transporters are involved in a wide range of processes including apoptotic cell corpse removal (Wu and Horvitz 1998), dauer formation (Yabe et al 2005), RNAi (Sundaram et al 2006b), directed sperm motility (Kubagawa et al 2006), as well as resistance to toxins (Broeks et al 1995), heavy metals (Broeks et al 1996;Vatamaniuk et al 2005), and pathogens (Mahajan-Miklos et al 1999).…”

TheCaenorhabditis elegans rsd-2andrsd-6Genes Are Required for Chromosome Functions During Exposure to Unfavorable Environments

“…Feeding-based assays for RNAi defects: ''Feeding plates'' harboring bacteria engineered to express dsRNA were prepared using HT115(DE3) host bacteria as described (Timmons et al 2001;Hull and Timmons 2004;Sundaram et al 2006). HT115(DE3) strains harboring a pop-1 cDNA in L4440 (pop-1 food) produce sterility in young animals; unc-22 food induces a twitching phenotype.…”

“…We mapped the alleles using standard genetic mapping techniques and pop-1 food to display RNAi defects in mutant homozygotes as described (Sundaram et al 2006).…”

“…Animals were then washed in PBS/3% BSA, blocked in 3% BSA for 1 hr, incubated at 4°in primary antibody diluted in 3% BSA overnight, washed in 3% BSA for three to five washes, and incubated in secondary antibody for at least 2 hr at room temperature. The anti-HAF-6 antibody was purified over a HAF-6TGST affinity column and used at 1:100 dilution or undiluted, depending upon the fraction (Sundaram et al 2006). Anticalreticulin antibodies were a gift from Joohong Ahnn (Park et al 2001) and were used at 1:200 or 1:500 dilution.…”