Characterization of SID-1-dependent and independent intergenerational RNA transport pathways in Caenorhabditis elegans

Systemic RNA interference (RNAi) in C. elegans is dependent on sid-1 (WO_SCPLOWINSTONC_SCPLOW et al. 2002), sid-3 (JO_SCPLOWOSEC_SCPLOW et al. 2012) and sid-5 (HO_SCPLOWINASC_SCPLOW et al. 2012). After injection, expression, or ingestion, double-stranded RNA (dsRNA) is transported between cells throughout the animal to enable RNAi in most tissues, including the germline and progeny. Here, we characterize the role of the Sid genes in transport of dsRNA to progeny. We previously reported that dsRNA injected directly in the germline unexpectedly requires sid-1 activity in the progeny to initiate RNAi (WO_SCPLOWINSTONC_SCPLOW et al. 2002). We now show that germline injected dsRNA can travel by three independent pathways to silence gene expression in embryos. First, germline injected dsRNA is delivered, presumably by bulk flow, into oocytes and embryos. This means of delivery, which does not require sid-1, is limited by the amount and location of injected dsRNA. Second, maternal sid-1 transports extracellular dsRNA into the germline where it can silence maternal deposited mRNAs and segregate to embryos to silence embryonically expressed mRNAs. Third, extracellular dsRNA is also endocytosed into oocytes by the low-density lipoprotein (LDL) receptor superfamily homolog RME-2. The endocytosed dsRNA then requires sid-1 and sid-5 in embryos to silence embryonically expressed genes. Extracellular fluorescent dsRNA, once endocytosed into oocytes, does not co-localize with VIT2::GFP and it does not require sid-1 activity to segregate from the late endocytosis marker GFP::RAB-7 in early embryos. In conclusion, we identify genes and pathways that function redundantly for intergenerational RNA transfer that may represent mechanisms for transgenerational epigenetic inheritance.