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Characterization of a TUTase/RNase complex required for Drosophila gametogenesis.

ABSTRACT: Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce Drosophila as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure-function analysis of the Drosophila TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of Tailor and Dis3L2, which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis.

SUBMITTER: Lin CJ

PROVIDER: S-EPMC5311484 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Characterization of a TUTase/RNase complex required for <i>Drosophila</i> gametogenesis.

Lin Ching-Jung CJ Wen Jiayu J Bejarano Fernando F Hu Fuqu F Bortolamiol-Becet Diane D Kan Lijuan L Sanfilippo Piero P Kondo Shu S Lai Eric C EC

RNA (New York, N.Y.) 20161214 3

Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cel ...[more]

PMID: 27974621

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Project description:Ribonucleases belonging to the RNase T2 family are enzymes associated with the secretory pathway that are almost absolutely conserved in all eukaryotes. Studies in plants and vertebrates suggest they have an important housekeeping function in rRNA recycling. However, little is known about this family of enzymes in protostomes. We characterized RNase X25, the only RNase T2 enzyme in Drosophila melanogaster. We found that RNase X25 is the major contributor of ribonuclease activity in flies as detected by in gel assays, and has an acidic pH preference. Gene expression analyses showed that the RNase X25 transcript is present in all adult tissues and developmental stages. RNase X25 expression is elevated in response to nutritional stresses; consistent with the hypothesis that this enzyme has a housekeeping role in recycling RNA. A correlation between induction of RNase X25 expression and autophagy was observed. Moreover, induction of gene expression was triggered by oxidative stress suggesting that RNase X25 may have additional roles in stress responses. Phylogenetic analyses of this family in protostomes showed that RNase T2 genes have undergone duplication events followed by divergence in several phyla, including the loss of catalytic residues, and suggest that RNase T2 proteins have acquired novel functions. Among those, it is likely that a role in host immunosuppression evolved independently in several groups, including parasitic Platyhelminthes and parasitoid wasps. The presence of only one RNase T2 gene in the D. melanogaster genome, without any other evident secretory RNase activity detected, makes this organism an ideal system to study the cellular functions of RNase T2 proteins associated with RNA recycling and maintenance of cellular homeostasis. On the other hand, the discovery of gene duplications in several protostome genomes also presents interesting new avenues to study additional biological functions of this ancient family of proteins.

Dataset Information

Characterization of a TUTase/RNase complex required for Drosophila gametogenesis.

Publications

Characterization of a TUTase/RNase complex required for <i>Drosophila</i> gametogenesis.

Similar Datasets

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