DsRNase1 Contribution to DsRNA Degradation Activity in the Sf9 Cells Conditioned Medium

Overview

Journal Front Insect Sci

Date 2024 Mar 12

PMID 38469530

Authors

Jinmo Koo

Subba Reddy Palli

Affiliations

Soon will be listed here.

Abstract

RNA interference (RNAi) is inefficient in lepidopteran insects, including . RNase activity in the lumen and hemocoel is known to contribute to low RNAi efficiency in these insects. Conditioned medium from Sf9 cells developed from ovaries of shows high dsRNA degradation activity. But the enzymes responsible for this activity have not been identified. The nuclease genes that are highly expressed in Sf9 cells, REase, RNaseT2, and dsRNase1, were identified. Knockdown of dsRNase1 in Sf9 cells resulted in a reduction of dsRNA degradation activity in the Sf9 cells conditioned medium. Knockdown of dsRNase1 also increased RNAi efficiency in Sf9 cells. The results from these studies identified a major player in dsRNA degradation activity in the Sf9 cells conditioned medium. We also describe an efficient system that can be used to identify other genes responsible for dsRNA degradation and RNAi efficiency in Sf9 cells.

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References

Kolliopoulou A, Kontogiannatos D, Mazurek A, Prifti I, Christopoulou V, Labropoulou V . Analysis of dsRNA production during baculovirus infection of Hi5 cells: RNA hairpins expressed by very late promoters do not trigger gene silencing. Front Insect Sci. 2024; 2:959077. PMC: 10926400. DOI: 10.3389/finsc.2022.959077. View

Dhandapani R, Gurusamy D, Palli S . Development of Catechin, Poly-l-lysine, and Double-Stranded RNA Nanoparticles. ACS Appl Bio Mater. 2022; 4(5):4310-4318. DOI: 10.1021/acsabm.1c00109. View

Chen X, Chereddy S, Gurusamy D, Palli S . Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda. Insect Biochem Mol Biol. 2020; 126:103455. DOI: 10.1016/j.ibmb.2020.103455. View

Gurusamy D, Mogilicherla K, Shukla J, Palli S . Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda. Arch Insect Biochem Physiol. 2020; 104(4):e21678. DOI: 10.1002/arch.21678. View

Winston W, Molodowitch C, Hunter C . Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science. 2002; 295(5564):2456-9. DOI: 10.1126/science.1068836. View

Yoon J, Gurusamy D, Palli S . Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda. Insect Biochem Mol Biol. 2017; 90:53-60. DOI: 10.1016/j.ibmb.2017.09.011. View

Cooper A, Song H, Shi X, Yu Z, Lorenzen M, Silver K . Molecular Characterizations of Double-Stranded RNA Degrading Nuclease Genes from . Insects. 2020; 11(10). PMC: 7598268. DOI: 10.3390/insects11100652. View

Guan R, Li H, Fan Y, Hu S, Christiaens O, Smagghe G . A nuclease specific to lepidopteran insects suppresses RNAi. J Biol Chem. 2018; 293(16):6011-6021. PMC: 5912458. DOI: 10.1074/jbc.RA117.001553. View

Almeida Garcia R, Macedo L, Cabral do Nascimento D, Gillet F, Moreira-Pinto C, Faheem M . Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis. PLoS One. 2017; 12(12):e0189600. PMC: 5738047. DOI: 10.1371/journal.pone.0189600. View

10.

Dow . pH GRADIENTS IN LEPIDOPTERAN MIDGUT. J Exp Biol. 1992; 172(Pt 1):355-375. DOI: 10.1242/jeb.172.1.355. View

11.

Chen X, Koo J, Gurusamy D, Mogilicherla K, Palli S . systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner. RNA Biol. 2020; 18(9):1291-1299. PMC: 8354605. DOI: 10.1080/15476286.2020.1842632. View

12.

Song H, Fan Y, Zhang J, Cooper A, Silver K, Li D . Contributions of dsRNases to differential RNAi efficiencies between the injection and oral delivery of dsRNA in Locusta migratoria. Pest Manag Sci. 2018; 75(6):1707-1717. DOI: 10.1002/ps.5291. View

13.

Yoon J, Shukla J, Gong Z, Mogilicherla K, Palli S . RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors. Insect Biochem Mol Biol. 2016; 78:78-88. DOI: 10.1016/j.ibmb.2016.09.002. View

14.

Song H, Zhang J, Li D, Cooper A, Silver K, Li T . A double-stranded RNA degrading enzyme reduces the efficiency of oral RNA interference in migratory locust. Insect Biochem Mol Biol. 2017; 86:68-80. DOI: 10.1016/j.ibmb.2017.05.008. View

15.

Shukla J, Kalsi M, Sethi A, Narva K, Fishilevich E, Singh S . Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects. RNA Biol. 2016; 13(7):656-69. PMC: 4962799. DOI: 10.1080/15476286.2016.1191728. View

16.

Gurusamy D, Mogilicherla K, Palli S . Chitosan nanoparticles help double-stranded RNA escape from endosomes and improve RNA interference in the fall armyworm, Spodoptera frugiperda. Arch Insect Biochem Physiol. 2020; 104(4):e21677. DOI: 10.1002/arch.21677. View

17.

Singh I, Singh S, Mogilicherla K, Shukla J, Palli S . Comparative analysis of double-stranded RNA degradation and processing in insects. Sci Rep. 2017; 7(1):17059. PMC: 5719073. DOI: 10.1038/s41598-017-17134-2. View

18.

Peng Y, Wang K, Zhu G, Han Q, Chen J, Elzaki M . Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae). Pestic Biochem Physiol. 2019; 162:86-95. DOI: 10.1016/j.pestbp.2019.09.011. View

19.

Burns B, Mendz G, Hazell S . Methods for the Measurement of a Bacterial Enzyme Activity in Cell Lysates and Extracts. Biol Proced Online. 2003; 1:17-26. PMC: 140121. DOI: 10.1251/bpo5. View

20.

Prentice K, Smagghe G, Gheysen G, Christiaens O . Nuclease activity decreases the RNAi response in the sweetpotato weevil Cylas puncticollis. Insect Biochem Mol Biol. 2019; 110:80-89. DOI: 10.1016/j.ibmb.2019.04.001. View