The endogenous piRNA propiR1 has two isoforms that differentially associate with Piwi4 and Piwi5. (A) Ranked list of the top 25 most abundant piRNAs in Piwi5 immunoprecipitation (IP) in Aag2 cells. The heatmap (left) represents piRNA-enrichment in different PIWI protein IP libraries; the bar chart (right) shows piRNA abundance in Aag2 cells. Dark gray bars indicate transposon-derived piRNAs; light gray bars indicate piRNAs not mapping to annotated transposons. (B) Enrichment of the two most abundant propiR1 isoforms (27 and 30 nt) in small RNA sequencing libraries generated from endogenous PIWI protein IPs. (C) Northern blot analysis of propiR1 enrichment in PIWI protein IPs using antibodies targeting endogenous PIWI proteins from Aag2 cell lysates. An IP in which the antibody was omitted serves as control (No ab). (D) Northern blot analysis of propiR1 and miR-2940-3p in RNA samples subjected to β-elimination as indicated. The blot was reused from Halbach et al. (2020) and reprobed for propiR1. The miR2940-3p panel is shown here again as a control (reproduced with permission from Fig. 1B in Halbach et al. 2020, © Springer Nature). (E) Northern blot analysis of propiR1 in Aag2 cells transfected with dsRNA targeting the indicated genes, or Renilla luciferase (dsRen) as a control. U6 snRNA serves as loading control. Knockdown efficiencies are shown in Supplemental Figure S1C. (F) Northern blot analysis of propiR1 in Piwi4 IP material from Aag2 cells treated with Piwi5 or control dsRNA (Luciferase, dsLuc), and in Piwi5 IP material treated with Piwi4 and control dsRNA. rRNA serves as loading control. (G) Schematic representation of propiR1 sites on chromosome 3 of Ae. aegypti and their piRNA coverage. propiR1 sites are indicated in red (top row). Protein-coding and noncoding genes, pseudogenes and piRNA clusters (as annotated in VectorBase) in a 200 kb window flanking the propiR1 piRNA sites are color-coded as indicated. piRNA clusters (in blue) were named according to chromosomal location, as described in Crava et al. (2021). VectorBase gene identifiers (without the AAEL0 species prefix) were used to refer to transcripts. Small RNA (19–32 nt) coverage in Aag2 cells is indicated relative to 106 miRNAs. (H) Sequence conservation between the seven propiR1 sites (marked as a gray box) and 500 bp flanking regions. The conservation scores (solid purple line) and 50% confidence intervals (gray shading) are indicated for each nucleotide position.
