Studies in C. elegans have shown that dsRNA expressed in neurons can transmit a signal to the germline of an animal to cause silencing of a germline gene in that animal and in its descendants. The molecular details of how mobile RNA is processed, exported, and transmitted from neurons to the germline are poorly understood. Tissue-specific rescue experiments using repetitive transgenes suggest that MUT-2, a putative RNA nucleotidyltransferase, is sufficient in the sending cells for gene silencing by mobile dsRNA. One hypothesis suggested by this finding is that forms of RNA that are modified by MUT-2 move between cells. Alternatively, use of repetitive transgenes could have led to misexpression in receiving cells and MUT-2 might function only in receiving cells. Distinguishing between these possibilities requires the development of better reagents and methods for the analysis of MUT-2 and small RNAs. In the absence of MUT-2, transposons are activated, leading to progressive mutagenesis in the background, which could complicate interpretations. Indeed, when we generated new deletions of
mut-2 using CRISPR-based genome editing and analyzed three independently propagated
mut-2(-) deletion lines, we did not detect the previously reported reduced brood size. To examine possible changes in small RNAs that result from loss of
mut-2, we have developed a sensitive northern blotting approach that can detect RNAs as short as 14 nt and thus effectively complement RNA-seq (1). Finally, we are defining the neurons that can export dsRNA to elicit the most effective and enduring transgenerational gene silencing through a large-scale effort called the Transgenerational Brain Initiative using cohorts of ~35 undergraduates each year. This effort will create and characterize a collection of strains expressing dsRNA, reporter genes, and MUT-2 from single copy transgenes in different subsets of neurons. Collectively, with these tools, we will establish where MUT-2 acts to initiate transgenerational gene silencing triggered by neuronal dsRNA. 1. Choi et al., Nucleic Acids Research, 2017.