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

Abstract

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.

Making Invisible RNA Visible: Discriminative Sequencing Methods for RNA Molecules with Specific Terminal Formations

“…Infection-induced tRNA halves could function as immune boosters by activating endosomal Toll-like receptor 7 [38]. tRNA halves can further serve as direct precursors for shorter sncRNAs such as piRNAs [43,44]. Because ANG-medicated cleavage leaves a 2 ,3 -cP and 5 -P in 5 -and 3 -cleavage products [45], respectively, 5 -tRNA halves contain a 5 -P (from mature tRNAs) and a 2 ,3 -cP, while 3 -tRNA halves possess a 5 -OH and an amino acid (AA) at the 3 -end (from mature tRNAs) (Figure 1B), which has been experimentally validated [37].…”

“…Although Dicer and ANG are involved in the biogenesis of some tRFs [48,49], detailed regulatory mechanisms and other biogenesis factors for tRFs remain elusive, designating tRFs as the molecules whose terminal structures have not yet been well defined. The tRFs that function as miRNAs or piRNAs by binding to AGO or PIWI proteins [32,43,44] should contain the 5 -P/3 -OH or 5 -P/2 -O-Me ends, respectively. tRFs further have a variety of functions, such as regulating gene expression via modulation of mRNA stability or translation, preventing cell apoptosis, and promoting virus infection, and their dysregulations are involved in various diseases [31,33,34,50].…”

“…Even if the 3 -AD ligation to the 2 -O-Me end is inefficient, the enrichment step enables amplifying and sequencing 2 -O-Me-containing RNAs as major species (Figure 1C). The combination of periodate oxidation and RNA-seq has been used for the sequencings of 2 -O-Me-containing RNAs, mainly for animal piRNAs [44,69,70].…”

“…However, the population of the 2 -O-Me-containing RNAs in cP-RNA-seq data is expected to be negligibly minor due to inefficient ligation of 3 -AD to the 2 -O-Me end. The cP-RNA-seq has been used to sequence 2 ,3 -cP-containing tRNA halves in cancer cells [37,47], immune cells [38], and germ cells [43,44]. The first genome-wide identification of 2 ,3 -cP-containing sncRNAs revealed numerous mRNAand rRNA-derived 2 ,3 -cP-containing sncRNAs, as well as tRNA halves, in various mouse tissues [46].…”

Mammalian antiviral systems directed by small RNA

“…Several tRFs are reported to bind Piwil4 (Hiwi2) in both normal and transformed human cells [ 127 ]. PIWIL2 (HILI) has been reported to interact with intact tRNAs and induce translational repression of HIV-1 via this activity [ 128 ], though HILI may also interact with tRFs that were undetected using the methods applied [ 129 ].…”