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. 2017 Jan;23(1):86–97. doi: 10.1261/rna.058875.116

FIGURE 5.

FIGURE 5.

Artificial piRNA production from the silkworm Actin A3 gene. (A,B) The artificial piRNA production system targeting the silkworm Actin A3 gene. BmN-4 cells are transfected with plasmids expressing pre-piRNA-A (A) or pre-piRNA-A-nc (B). The pre-piRNA-A mRNA is cleaved by the Masc piRNA–BmAgo3 complex, whereas pre-piRNA-A-nc mRNA is not. The resulting 3′ pre-piRNA-A fragment is loaded into Siwi, thus producing the piRNA-A–Siwi complex, which targets silkworm Actin A3 mRNA. The cleaved 3′ fragment is then loaded into another PIWI protein, BmAgo3, thereby producing the piRNA-B–BmAgo3 complex. (C,D) Normalized piRNA-A (C) and piRNA-B (D) reads in total piRNA libraries from BmN-4 cells transfected with plasmids expressing pre-piRNA-A or pre-piRNA-A-nc. Mapping reads against 1811 transposons were used for normalization. (E,F) Artificial piRNAs derived from the Actin A3 gene. The start sites of piRNAs mapped to the Actin A3 gene are indicated. (G) Expression levels of Actin A3 mRNA. Expression of Actin A3 mRNA was examined by RT-qPCR. Data shown are mean ± standard deviation (n = 5). Data were subjected to Student's t-test (one-sided). (*) P < 0.05 (P = 0.02046).