Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata
Abstract
:1. Introduction
1.1. RNAi and Its Use in Decapods
1.2. Known Mechanisms of Systemic RNAi—A Drop in the Ocean
1.3. dsRNA Transport in Serum
2. Results
2.1. Correlation between Humoral Response to dsRNA and Silencing Capacity Using Exogenous dsRNA Across Phyla
2.2. Identification of Putative dsRNA Binding Protein Complex in Sera via Mass Spectrometry Analysis
2.3. Following the Gel Shift in Crustaceans
2.4. dsRNA-Induced IAG Knockdown in T. australiensis
3. Discussion
4. Materials and Methods
4.1. Hemolymph, Hemocoel, Plasma, and Serum Collection
4.2. Double-Stranded RNA Production
4.3. Electromobility Shift Assay (EMSA)
4.4. uHPLC Tandom QTof Mass Spectrometry Analysis
4.5. In Vivo T. Australiensis IAG Silencing
4.6. RNA Extraction and RT qPCR
4.7. Bioinformatic Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Species | Proteins Identified (via BlastP) | No. of Peptides | Sequence Coverage | e-Value | % Identity |
---|---|---|---|---|---|
C. quadricarinatus | clotting protein precursor [Pacifastacus leniusculus] | 1 | 1 | 0 | 50% |
E. lanceolatus | apolipoprotein A-Ib [Epinephelus lanceolatus] | 10 | 24 | 0 | 99.62% |
E. lanceolatus | 14 kDa apolipoprotein, partial [Epinephelus bruneus] | 7 | 47 | 1 × 10−87 | 89.51 |
B. mori | apolipophorin III precursor [Bombyx mori] | 2 | 14 | 7 × 10−131 | 100% |
B. mori | low molecular 30 kDa lipoprotein PBMHP-6 [Bombyx mandarina] | 1 | 4 | 0 | 97.66% |
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Banks, T.M.; Wang, T.; Fitzgibbon, Q.P.; Smith, G.G.; Ventura, T. Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata. Int. J. Mol. Sci. 2020, 21, 6967. https://doi.org/10.3390/ijms21186967
Banks TM, Wang T, Fitzgibbon QP, Smith GG, Ventura T. Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata. International Journal of Molecular Sciences. 2020; 21(18):6967. https://doi.org/10.3390/ijms21186967
Chicago/Turabian StyleBanks, Thomas M., Tianfang Wang, Quinn P. Fitzgibbon, Gregory G. Smith, and Tomer Ventura. 2020. "Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata" International Journal of Molecular Sciences 21, no. 18: 6967. https://doi.org/10.3390/ijms21186967
APA StyleBanks, T. M., Wang, T., Fitzgibbon, Q. P., Smith, G. G., & Ventura, T. (2020). Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla—Towards Finding the Missing Link in Achelata. International Journal of Molecular Sciences, 21(18), 6967. https://doi.org/10.3390/ijms21186967