Lack of GTSF1 results in a target RNA (pit‐RNA) unsliced at the cleavage site for MILI‐directed secondary piRNA processing Lack of GTSF1 results in a target RNA (pit‐RNA) unsliced at the cleavage site for MILI‐directed secondary piRNA processing Schematic illustration of the modified RACE method.Modified RACE was used for the detection of pit‐RNA cleaved by MILI. Arrow shows a specific band amplified by nested PCRs (417 bp). Arrowhead shows nonspecific bands. Amplified DNAs were separately purified and sequenced. The same results were obtained in biologically duplicated samples.RT–qPCR analysis of pit‐RNA expression in Gtsf1+/− and Gtsf1−/− E17.5 testes. Pit‐RNA expression was normalized to Gtsf1l expression [48] and plotted relative to its average expression level in Gtsf1+/− testes. Error bars represent SD (n = 3). *P < 0.01, Student's t‐test.Model of involvement of mouse GTSF1 in the prenatal piRNA pathway. Our data suggest that mouse GTSF1 has crucial role(s) in the step where the PIWI‐piRNA complex grasps and stabilizes a target RNA. Red and blue arrows represent sense and antisense RNAs (including piRNAs, its precursors, or source RNAs), respectively. We propose that mouse GTSF1 is involved in not only posttranscriptional gene silencing (PTGS) with MILI but also transcriptional gene silencing (TGS) with MIWI2. Takuji Yoshimura et al. EMBO Rep. 2018;19:e42054 © as stated in the article, figure or figure legend