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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.

SUBMITTER: Shigematsu M

PROVIDER: S-EPMC8302750 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3'-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3'-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3'-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3'-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

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

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