Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway

<p>RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn <i>Macrobrachium rosenbergii</i> by knocking down the transcript level of an insulin-like androgenic gland hormone (<i>Mr-IAG</i>) through injections of dsRNA of the entire <i>Mr-IAG</i> ORF sequence (ds<i>Mr-IAG</i> – 518bp). Interestingly, <i>in-vivo</i> knockdown success and ds<i>Mr-IAG</i> lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in <i>M. rosenbergii</i>. We discovered the <i>Mr-Dicer</i> and <i>Mr-Argonaute</i> gene families, associated with the major knockdown pathways, in our <i>M. rosenbergii</i> transcriptomic library. In response to ds<i>Mr-IAG</i> administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a <i>SID1</i> gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing <i>Mr-SID1</i> specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments <i>in-vitro</i>, the case presented here demonstrates length dependency <i>in-vivo</i>, suggesting further complexity in the context of the entire organism.</p