FIGURE 3.

RNAi knockdowns of genes encoding TUDOR domain-containing proteins identified BmPAPI as a factor affecting piRNA length. (A) The indicated genes encoding TUDOR domain-containing proteins, PIWI (positive control), and Renilla luciferase (Rluc, negative control) were silenced by RNAi in BmN4 cells. Total RNAs from the cells were subjected to Northern blots against piRNA-1 (SIWI-bound), piRNA-2 (BmAGO3-bound), and let-7 miRNA (control). Quantifications of mRNA to confirm knockdown efficiencies are shown in Supplemental Figure S2. piRNAs that were extended as a result of BmPAPI knockdown are indicated with red lines. (B) Total cell lysates from BmN4 cells treated with dsRNAs targeting Rluc (control), SIWI, BmAGO3, and BmPAPI were subjected to Western blots with the indicated antibodies. (C) Total RNAs from Rluc- (control) and BmPAPI-silenced BmN4 cells were treated with NaIO4, subjected to β-elimination, and detected by Northern blots. Total RNA treated with phosphatase before the NaIO4 reaction was also analyzed. Let-7 was shortened by β-elimination due to its hydroxyl 3′ terminus, whereas piRNAs were not affected due to 2′-O-methylation at their 3′ termini. (D) Immunofluorescence staining of endogenous SIWI and BmAGO3 (green) and DNA (blue) in Rluc- or BmPAPI-silenced BmN4 cells.