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

Thomas M Banks,Quinn P Fitzgibbon,Tianfang Wang,Gregory G Smith,Tomer Ventura

doi:10.3390/ijms21186967

Thomas M Banks, Quinn P Fitzgibbon + Show 3 more

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https://doi.org/10.3390/ijms21186967

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Abstract

RNA interference (RNAi) has become a widely utilized method for studying gene function, yet despite this many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double-stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi and may be the limiting factor in RNAi capacity for species such as spiny lobsters, where gene silencing is not functional. Incubating sera from a variety of species across phyla with dsRNA led to a gel mobility shift in species in which systemic RNAi has been observed, with this response being absent in species in which systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters and for a better understanding of the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

Highlights

In 1998, Fire et al demonstrated potent and efficient gene silencing in the nematode Caenorhabditis elegans, which built on the phenomenon of antisense RNA technology [1]
An electromobility shift assay (EMSA) revealed that 1 μg of double-stranded RNA (dsRNA) pre-incubated with 10 μL of serum from prawn species P. monodon, M. rosenbergii, and crayfish species C. quadricarinatus migrated slower than non-incubated dsRNA, with a retarded band of slower mobility than the 8 kb DNA marker, compared to the 660 bp band of the lone dsRNA (Figure 2)
The serum from three spiny lobster species, S. verreauxi, P. ornatus, and Panulirus homarus, did not affect the migration of dsRNA (Figure 2). This gel shift correlates with in vivo dsRNA-induced RNA interference (RNAi), which has been observed in C. quadricarinatus, P. monodon, and M. rosenbergii, but not in any spiny lobster species to date [10,12,40]

Summary

Introduction

In 1998, Fire et al demonstrated potent and efficient gene silencing in the nematode Caenorhabditis elegans, which built on the phenomenon of antisense RNA technology [1]. Injection of sense and antisense RNA for the myofilament protein coding gene unc-22, which formed double-stranded RNA (dsRNA), showed the twitching phenotype associated with unc-22 knockdown [1]. The use of exogenous dsRNA to elicit gene silencing has become known as RNA interference (RNAi) and has developed into a widely used molecular tool to study gene function [2]. Apart from being a useful method to study gene function, RNAi holds great potential in biotechnology, in areas such as pest management, molecular therapeutics, and aquaculture [2,3]. The exogenous small interfering RNA pathway (exo-siRNA), which is associated with exogenous dsRNA-induced gene silencing, is a naturally occurring mechanism with roles in innate antiviral immunity and defence from mobile genetic elements [4] (Figure 1).

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