FIGURE 1.

Products of target RNA slicing remain bound to PIWI complexes in mouse testes. (A) Perfect or bulge piR-A target sites were inserted into the last exon of Ythdc2. Single or 10 tandem sites with spacers were inserted. MILI and MIWI loaded sequences were identified by sequencing of immunoprecipitated RNAs. (B) Perfect (Perf) piR-A sites were designed to be fully complementary to endogenous piR-A piRNA. The bulge piR-A sites were designed to contain noncomplementary bases to piR-A nucleotides at positions 10 and 11. (C) Length profiles of immunoprecipitated small RNAs from 1xPerf mouse. (D) The position (=distance from 5′ nt of targeting piR-A) and abundance of 5′ ends of reads produced from reporter target sites. In the case of 10xPerf, the metaplot summarizes the read counts from all ten sites. Red triangle refers to expected position of MILI/MIWI cleavage guided by piR-A. (E) Schematic overview of RNA fragments identified in MILI/MIWI immunoprecipitations from 10xPerf mice. (F) Coverage of reads is shown separately for RNAs with their 5′ end starting at a specific distance from the first piR-A target nucleotide (=0 distance). These RNAs are of specific lengths, with the 3′ ends always created by MILI/MIWI slicing at a subsequent site. Metaplots summarize the read counts from 10 sites. (G) The RNAs produced from sites 7 and 8 in 10xPerf and 10xBulge are shown together with their abundance. Reads from MILI and MIWI immunoprecipitations are combined. Only reads with abundance ≥0.05 rpm are shown.