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

Nir Shpak, Rivka Manor, Lihie Katzir Abilevich, Ortal Mantal, Keshet Shavit, Eliahu D. Aflalo, Debra Toiber, Amir Sagi

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

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.

Original languageEnglish
Pages (from-to)1766-1775
Number of pages10
JournalRNA Biology
Volume14
Issue number12
DOIs
StatePublished - 2 Dec 2017

Keywords

  • Argonaute
  • Crustacea
  • Dicer
  • M. rosenbergii insulin-like androgenic hormone (Mr-IAG)
  • Macrobrachium rosenbergii
  • SID1
  • dsRNA
  • systemic RNAi

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