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2. Search for limiting factors in the RNAi pathway in silkmoth tissues and the silkmoth-derived Bm5 cell line : the RNA-binding proteins R2D2 and Translin

Author

Swevers, Luc, Liu, Jisheng, Huvenne, Hanneke, and Smagghe, Guy

Subjects
EXPRESSION, GENOME, INTERFERENCE, DROSOPHILA, GENES, SILKWORM BOMBYX-MORI, fungi, INSECTS, Biology and Life Sciences, TRIBOLIUM-CASTANEUM, DOUBLE-STRANDED-RNA, NUCLEAR POLYHEDROSIS-VIRUS
Abstract

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

Published
2011

3. RNA interference in Lepidoptera : An overview of successful and unsuccessful studies and implications for experimental design

Author

Terenius, Olle, Papanicolaou, Alexie, Garbutt, Jennie S., Eleftherianos, Ioannis, Huvenne, Hanneke, Kanginakudru, Sriramana, Albrechtsen, Merete, An, Chunju, Aymeric, Jean-Luc, Barthel, Andrea, Bebas, Piotr, Bitra, Kavita, Bravo, Alejandra, Chevalier, François, Collinge, Derek P., Crava, Cristina M., de Maagd, Ruud A., Duvic, Bernard, Erlandson, Martin, Faye, Ingrid, Felföldi, Gabriella, Fujiwara, Haruhiko, Futahashi, Ryo, Gandhe, Archana S., Gatehouse, Heather S., Gatehouse, Laurence N., Giebultowicz, Jadwiga M., Gómez, Isabel, Grimmelikhuijzen, Cornelis J. P., Groot, Astrid T., Hauser, Frank, Heckel, David G., Hegedus, Dwayne D., Hrycaj, Steven, Huang, Lihua, Hull, J. Joe, Iatrou, Kostas, Iga, Masatoshi, Kanost, Michael R., Kotwica, Joanna, Li, Changyou, Li, Jianghong, Liu, Jisheng, Lundmark, Magnus, Matsumoto, Shogo, Meyering-Vos, Martina, Millichap, Peter J., Monteiro, Antónia, Mrinal, Nirotpal, Niimi, Teruyuki, Nowara, Daniela, Ohnishi, Atsushi, Oostra, Vicencio, Ozaki, Katsuhisa, Papakonstantinou, Maria, Popadic, Aleksandar, Rajam, Manchikatla V., Saenko, Suzanne, Simpson, Robert M., Soberón, Mario, Strand, Michael R., Tomita, Shuichiro, Toprak, Umut, Wang, Ping, Wee, Choon Wei, Whyard, Steven, Zhang, Wenqing, Nagaraju, Javaregowda, Ffrench-Constant, Richard H., Herrero, Salvador, Gordon, Karl, Swevers, Luc, Smagghe, Guy, Terenius, Olle, Papanicolaou, Alexie, Garbutt, Jennie S., Eleftherianos, Ioannis, Huvenne, Hanneke, Kanginakudru, Sriramana, Albrechtsen, Merete, An, Chunju, Aymeric, Jean-Luc, Barthel, Andrea, Bebas, Piotr, Bitra, Kavita, Bravo, Alejandra, Chevalier, François, Collinge, Derek P., Crava, Cristina M., de Maagd, Ruud A., Duvic, Bernard, Erlandson, Martin, Faye, Ingrid, Felföldi, Gabriella, Fujiwara, Haruhiko, Futahashi, Ryo, Gandhe, Archana S., Gatehouse, Heather S., Gatehouse, Laurence N., Giebultowicz, Jadwiga M., Gómez, Isabel, Grimmelikhuijzen, Cornelis J. P., Groot, Astrid T., Hauser, Frank, Heckel, David G., Hegedus, Dwayne D., Hrycaj, Steven, Huang, Lihua, Hull, J. Joe, Iatrou, Kostas, Iga, Masatoshi, Kanost, Michael R., Kotwica, Joanna, Li, Changyou, Li, Jianghong, Liu, Jisheng, Lundmark, Magnus, Matsumoto, Shogo, Meyering-Vos, Martina, Millichap, Peter J., Monteiro, Antónia, Mrinal, Nirotpal, Niimi, Teruyuki, Nowara, Daniela, Ohnishi, Atsushi, Oostra, Vicencio, Ozaki, Katsuhisa, Papakonstantinou, Maria, Popadic, Aleksandar, Rajam, Manchikatla V., Saenko, Suzanne, Simpson, Robert M., Soberón, Mario, Strand, Michael R., Tomita, Shuichiro, Toprak, Umut, Wang, Ping, Wee, Choon Wei, Whyard, Steven, Zhang, Wenqing, Nagaraju, Javaregowda, Ffrench-Constant, Richard H., Herrero, Salvador, Gordon, Karl, Swevers, Luc, and Smagghe, Guy

Abstract

Gene silencing through RNA interference (RNAi) has revolutionized the study of gene function, particularly in non-model insects. However, in Lepidoptera (moths and butterflies) RNAi has many times proven to be difficult to achieve. Most of the negative results have been anecdotal and the positive experiments have not been collected in such a way that they are possible to analyze. In this review, we have collected detailed data from more than 150 experiments including all to date published and many unpublished experiments. Despite a large variation in the data, trends that are found are that RNAi is particularly successful in the family Saturniidae and in genes involved in immunity. On the contrary, gene expression in epidermal tissues seems to be most difficult to silence. In addition, gene silencing by feeding dsRNA requires high concentrations for success. Possible causes for the variability of success in RNAi experiments in Lepidoptera are discussed. The review also points to a need to further investigate the mechanism of RNAi in lepidopteran insects and its possible connection to the innate immune response. Our general understanding of RNAi in Lepidoptera will be further aided in the future as our public database at http://insectacentral.org/RNAi will continue to gather information on RNAi experiments.

Published
2011
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