title: Oligoadenylation of 3' decay intermediates promotes cytoplasmic mRNA degradation in Drosophila cells.
abstract: Post-transcriptional 3' end addition of nucleotides is important in a variety of RNA decay pathways. We have examined the 3' end addition of nucleotides during the decay of the Hsp70 mRNA and a corresponding reporter RNA in Drosophila S2 cells by conventional sequencing of cDNAs obtained after mRNA circularization and by deep sequencing of dedicated libraries enriched for 3' decay intermediates along the length of the mRNA. Approximately 5%-10% of 3' decay intermediates carried nonencoded oligo(A) tails with a mean length of 2-3 nucleotides. RNAi experiments showed that the oligoadenylated RNA fragments were intermediates of exosomal decay and the noncanonical poly(A) polymerase Trf4-1 was mainly responsible for A addition. A hot spot of A addition corresponded to an intermediate of 3' decay that accumulated upon inhibition of decapping, and knockdown of Trf4-1 increased the abundance of this intermediate, suggesting that oligoadenylation facilitates 3' decay. Oligoadenylated 3' decay intermediates were found in the cytoplasmic fraction in association with ribosomes, and fluorescence microscopy revealed a cytoplasmic localization of Trf4-1. Thus, oligoadenylation enhances exosomal mRNA degradation in the cytoplasm.
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Harnisch
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Simona
authors.initials: S
authors.lastname: Cuzic-Feltens
authors.affiliation: Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria.
authors.forename: Juliane C
authors.initials: JC
authors.lastname: Dohm
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Götze
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190 Vienna, Austria.
authors.forename: Heinz
authors.initials: H
authors.lastname: Himmelbauer
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany.
authors.forename: Elmar
authors.initials: E
authors.lastname: Wahle
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004331:Drosophila melanogaster; D006868:Hydrolysis; D009843:Oligoribonucleotides / Q000378:metabolism*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D012333:RNA, Messenger; C023505:2',5'-oligoadenylate; D011062:Polynucleotide Adenylyltransferase
keywords: exosome; mRNA decay; noncanonical poly(A) polymerase; oligoadenylation