Nuclear RNAi Contributes to the Silencing of Off-target Genes and Repetitive Sequences in Caenorhabditis elegans. Caenorhabditis elegans

Small RNAs recognize, bind, and regulate other complementary cellular RNAs. The introduction of small RNAs to eukaryotic cells frequently results in unintended silencing of related, but not identical, RNAs: a process termed off-target gene silencing. Off-target gene silencing is one of the major concerns during the application of small RNA-based technologies for gene discovery and the treatment of human disease. Off-target gene silencing is commonly thought to be due to inherent biochemical limitations of the RNAi machinery. Here we show that, following the introduction of exogenous sources of dsRNA, the nuclear RNAi pathway, but not its cytoplasmic counterparts, is the primary source of off-target silencing in C. elegans. In addition, we show that during the normal course of growth and development the nuclear RNAi pathway regulates repetitive gene families. Therefore, we speculate that RNAi off-target effects might not be “mistakes” but, rather, an intentional and genetically programmed aspect of small RNA-mediated gene silencing, which might allow small RNAs to silence rapidly evolving parasitic nucleic acids. Finally, reducing off-target effects by manipulating the nuclear RNAi pathway in vivo might improve the efficacy of small RNA-based technologies. (The Zhou X, Xu F, Mao H, Ji J, Meng Y, Feng X, and Shouhong Guang (2014) Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans. Genetics 2014 May;197(1):121-32. doi: 10.1534/genetics.113.159780.) Overall design: To better understand the endogenous roles of the nuclear RNAi pathway, and to ask if nuclear RNAi drives off-target silencing during the normal course of growth and development, we immunoprecipitated NRDE-3 from the FLAG::GFP::NRDE-3 strain and deep sequenced the associated small RNAs from embryo lysates. eri-1(mg366);dpy-13(e458);FLAG::GFP::NRDE-3 fed dpy-13 RNAi was included as a control. We used a small RNA-cloning method compatible with cloning monophosphorylated and triphosphorylated small RNAs (i.e., 22G-RNAs). Small RNAs were isolated, pretreated with calf intestinal alkaline phosphatase to remove phosphate groups, treated with polynucleotide kinase to add a monophosphate group to the 5’-ends, and subjected to deep sequencing using the Illumina platform. Small RNAs were aligned to the C. elegans transcriptome (WS243 assembly) using Bowtie2 software with the default parameters, and the number of small RNAs targeting each gene was counted.