RNase κ promotes robust piRNA production by generating 2′,3′-cyclic phosphate-containing precursors

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.

(c) Siwi-or BmAgo3-bound piRNAs #2 identified in a previous study 5 were mapped to cP-RNAs or P-cP-RNAs and the obtained mapping ratios are shown. The results of Siwi-and BmAgo3-bound piRNAs #1 (identified in 6 ) are shown in Fig. 2a. (d) Among transposon-derived or rRNA-derived cP-RNAs/P-cP-RNAs, piRNA-mapped species were counted and their ratio to total species is shown.
(e, f) Scatter plots showing the correlations between the read numbers of Siwi-/BmAgo3-bound piRNAs (the data of #1 and #2 are shown in e and f, respectively) and cP-RNAs/P-cP-RNAs in 1,811 Bombyx transposons.
(g, h) Correlation of the terminal positions between Siwi-/BmAgo3-bound piRNAs (the data of #1 and #2 are shown in g and h, respectively) and their mapped cP-RNAs/P-cP-RNAs were analyzed, and matched rates from 5′-end (blue) and 3′-end (red) are shown.

Supplementary Figure 3. Analyses of BmN4 cP-RNAs and OH-RNAs
(a) Schematic representation of the procedures of OH-RNA-seq and cP-RNA-seq. In OH-RNAseq, RNAs are first 5′-phophorylated without affecting 3′-end by treatment with mutant T4 PNK, which lacks 3′-dephosphorylation activity, followed by AD ligation and cDNA amplification.
(b) BmN4 30-70-nt RNAs were gel-purified, mixed with spike-in control RNAs, and subjected to OH-RNA-seq and cP-RNA-seq. Amplified cDNAs were developed by native PAGE, followed by SYBR Gold staining. Both methods mainly amplified approximately 150-180-bp cDNA products. The cDNAs were not amplified by OH-RNA-seq with NaIO4 treatment or cP-RNA-seq with mutant T4 PNK treatment, suggesting successful cDNA amplification from respective targeted RNAs. The cDNA products, indicated by lines, were gel-purified and subjected to Illumina sequencing.
(d) Read length distributions of the obtained sequences from two biological replicates. Read numbers of OH-RNAs/cP-RNAs were normalized by those of spike-in RNAs.

Supplementary Figure 4. Analyses of mouse cP-RNAs and piRNAs
(a) RNAs from mouse testes were gel-purified and subjected to cP-RNA-seq and piRNA-seq (biological replicate #2).
(c) RNAs extracted from Mili-immunoprecipitates were subjected to P-cP-RNA-seq and piRNA-seq (biological replicate #2).   (c) BmN4 cell lysate was subjected to western blot using anti-BmRNase  antibody.      RNAs were subjected to northern blot for piR-a. Red and blue dots designate piR-a and its 3′end extended version, respectively.
(h) Total RNAs extracted from control-or BmRNase  KD cells were subjected to piRNA-seq.
Read length distributions of the transposon mapped piRNA reads are shown.  (e) Schematic representation of the enzymatic determination of 3′-end formation for the BmRNase -cleaved RNAs. After in vitro cleavage by BmRNase , the cleaved RNAs were treated with CIP or T4 PNK (NT: non-treated, a negative control) and subjected to 3′-AD ligation using T4 Rnl. The RNA-3′-AD ligation products were amplified by RT-qPCR and resolved in native PAGE.