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

Abstract

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 Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG - 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG 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 M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 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 in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata

“…SID-1 proteins first identified in C. elegans allow passive transport of dsRNA into the cell and are found ubiquitously in metazoan evolution, however whether they contribute to systemic RNAi seems to vary [18,19]. In crustaceans, however, SID-1 proteins in Macrobrachium rosenbergii and Litopenaeus vannamei were shown to facilitate dsRNA uptake [20][21][22]. Uptake of dsRNA may also occur via receptor-mediated endocytosis, as has been demonstrated in C. elegans and some insect species [17,[23][24][25].…”

Transcriptomic analysis of Macrobrachium rosenbergii (giant fresh water prawn) post-larvae in response to M. rosenbergii nodavirus (MrNV) infection: de novo assembly and functional annotation

“…In prawns, RNA interference (RNAi) plays a crucial role in antiviral immunity. Several key components of RNAi has been characterized in prawns including isoforms of dicer and argonaute in M. rosenbergii [90], L. vannamei [91–93], and P. monodon [94, 95]. Dicer, RNase III-like enzyme, is responsible for the cleavage of long dsRNA into 21–30 bp siRNA which initiates RNAi mechanism [96, 97].…”

Genes encoding putative bicarbonate transporters as a missing molecular link between molt and mineralization in crustaceans

“…The concentration of dsRNA was 5 µg per g body weight. This concentration of dsRNA was utilized since it is known to induce silencing in C. quadricarinatus 52 . The injections of dsRNA began at an MMI of ≈ 0.07 and were given daily over a period of 5 days.…”