title: An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.
abstract: N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.
authors.affiliation: Department of Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Ching-Jung
authors.initials: CJ
authors.lastname: Lin
authors.affiliation: Department of Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA Research School of Biological Sciences, The Australian National University, Acton ACT 2601, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Smibert
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Hu
authors.affiliation: Department of Biology, The City College of the City University of New York, New York 10031, USA The Graduate Center of the City University of New York, New York 10016, USA.
authors.forename: Johnny
authors.initials: J
authors.lastname: Ramroop
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Kellner
authors.affiliation: Research School of Biological Sciences, The Australian National University, Acton ACT 2601, Australia.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Benton
authors.affiliation: Department of Biology, The City College of the City University of New York, New York 10031, USA The Graduate Center of the City University of New York, New York 10016, USA.
authors.forename: Shubha
authors.initials: S
authors.lastname: Govind
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Rolf
authors.initials: R
authors.lastname: Sternglanz
authors.affiliation: Department of Developmental Biology, Sloan-Kettering Institute, New York, New York 10065, USA.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Lai
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D053898:Biosynthetic Pathways; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000502:physiology*; D004331:Drosophila melanogaster / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D005260:Female; D005816:Genetic Complementation Test; D060227:Imaginal Discs / Q000201:enzymology / Q000254:growth & development; D007814:Larva / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D008297:Male; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D009928:Organ Specificity; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; C003268:N(6)-(N-threonylcarbonyl)adenosine; D000241:Adenosine
keywords: Drosophila; Kae1; N6-threonylcarbamoyladenosine; t6A