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

Swevers L, Liu J, Huvenne H, Smagghe G - PLoS ONE (2011)

Bottom Line: 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.Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells.The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. swevers@bio.demokritos.gr

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.

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The sequences of BmTranslin isoforms.A. Amino-acid sequences of the two BmTranslin isoforms isolated from silkmoth tissues. The protein isoforms have different C-terminal ends (underlined). B. Comparison of the BmTranslin isoform sequences which shows the deletion in the BmTranslin1 sequence that causes a frameshift and a premature stop codon.
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pone-0020250-g002: The sequences of BmTranslin isoforms.A. Amino-acid sequences of the two BmTranslin isoforms isolated from silkmoth tissues. The protein isoforms have different C-terminal ends (underlined). B. Comparison of the BmTranslin isoform sequences which shows the deletion in the BmTranslin1 sequence that causes a frameshift and a premature stop codon.

Mentions: The expression pattern of factors of the small RNA pathways in Bm5 cells followed the general trend present in the silkmoth tissues (and includes absence of BmR2D2 mRNA; Figure 1), with the exception of the amplification of BmTranslin mRNA using primer pair 1 (Table 1; this primer pair is specific to the Genbank sequence NM_001046817; corresponding to “BmTranslin2”, see below) that did not result in a specific PCR product for the cell line. However, an alternative isoform of BmTranslin mRNA, with a truncation of the ORF at the C-terminus, “BmTranslin1”, was detected in both silkmoth tissues and Bm5 cells using primer pair 2 (Table 1). An EST of this isoform was also present in Silkbase (fdpeP14_F_N15.2). Figure 2A represents the complete amino-acid sequences of both the truncated isoform (“BmTranslin1”, amplified by primer pair 2 (Table 1), corresponding to EST fdpeP14_F_N15.2, and expressed in both silkmoth tissues and Bm5 cells) and the extended isoform (“BmTranslin2”, amplified by primer pair 3 (Table 1), corresponding to Genbank accession NM_001046817, and expressed in silkmoth tissues but not in Bm5 cells). It is noted that the conserved C-terminus present in Translin proteins in general [19] is not present in the BmTranslin1 isoform.


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

Swevers L, Liu J, Huvenne H, Smagghe G - PLoS ONE (2011)

The sequences of BmTranslin isoforms.A. Amino-acid sequences of the two BmTranslin isoforms isolated from silkmoth tissues. The protein isoforms have different C-terminal ends (underlined). B. Comparison of the BmTranslin isoform sequences which shows the deletion in the BmTranslin1 sequence that causes a frameshift and a premature stop codon.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102679&req=5

pone-0020250-g002: The sequences of BmTranslin isoforms.A. Amino-acid sequences of the two BmTranslin isoforms isolated from silkmoth tissues. The protein isoforms have different C-terminal ends (underlined). B. Comparison of the BmTranslin isoform sequences which shows the deletion in the BmTranslin1 sequence that causes a frameshift and a premature stop codon.
Mentions: The expression pattern of factors of the small RNA pathways in Bm5 cells followed the general trend present in the silkmoth tissues (and includes absence of BmR2D2 mRNA; Figure 1), with the exception of the amplification of BmTranslin mRNA using primer pair 1 (Table 1; this primer pair is specific to the Genbank sequence NM_001046817; corresponding to “BmTranslin2”, see below) that did not result in a specific PCR product for the cell line. However, an alternative isoform of BmTranslin mRNA, with a truncation of the ORF at the C-terminus, “BmTranslin1”, was detected in both silkmoth tissues and Bm5 cells using primer pair 2 (Table 1). An EST of this isoform was also present in Silkbase (fdpeP14_F_N15.2). Figure 2A represents the complete amino-acid sequences of both the truncated isoform (“BmTranslin1”, amplified by primer pair 2 (Table 1), corresponding to EST fdpeP14_F_N15.2, and expressed in both silkmoth tissues and Bm5 cells) and the extended isoform (“BmTranslin2”, amplified by primer pair 3 (Table 1), corresponding to Genbank accession NM_001046817, and expressed in silkmoth tissues but not in Bm5 cells). It is noted that the conserved C-terminus present in Translin proteins in general [19] is not present in the BmTranslin1 isoform.

Bottom Line: 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.Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells.The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed.

View Article: PubMed Central - PubMed

Affiliation: Insect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research Demokritos, Athens, Greece. swevers@bio.demokritos.gr

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.

Show MeSH
Related in: MedlinePlus