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Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

ABSTRACT: Drosophila RNase Z(L) (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3'-end processing. The biochemical function of RNase Z(L) is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZ(ED24) knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stages. We found that dRNaseZ activity is essential for all phases of fly development that involve cell division, including growth of adult tissue progenitors during larval and metamorphic stages, and gametogenesis in adults. At the cellular level, two major phenotypes were identified-cell growth deficiency in endoreplicating tissues and cell cycle arrest in mitotic tissues. While cell growth and proliferation are both dependant on protein synthesis, the two phenotypes displayed reliance on different dRNaseZ functions. We found that dRNaseZ KO completely blocks tRNA maturation without diminishing the abundance of mature tRNA molecules. Our data indicate that growth arrest of endoreplicating cells is primarily attributed to the relocation of the pool of mature tRNAs into the nuclei causing a decrease in translation efficiency. Mitotically dividing cells appear to be less dependent on translation machinery as they maintain their normal size when deprived of dRNaseZ activity, but rather display a cell cycle arrest at the G2-M transition.

SUBMITTER: Xie X

PROVIDER: S-EPMC3760340 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

Xie Xie X Dubrovskaya Veronica V Yacoub Nancy N Walska Joanna J Gleason Tara T Reid Katherine K Dubrovsky Edward B EB

Developmental biology 20130715 2

Drosophila RNase Z(L) (dRNaseZ) belongs to a family of endoribonucleases with a major role in tRNA 3'-end processing. The biochemical function of RNase Z(L) is conserved from yeast to human. Here we present a study of its biological function during Drosophila development. In flies, dRNaseZ provides a non-redundant function, as the RNZ(ED24) knockout (KO) mutation causes early larval lethality. Mosaic and conditional rescue techniques were employed to determine dRNaseZ requirements at later stage ...[more]

PMID: 23867108

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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.

Dataset Information

Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

Publications

Developmental roles of Drosophila tRNA processing endonuclease RNase ZL as revealed with a conditional rescue system.

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