Curled encodes the Drosophila homolog of the vertebrate circadian deadenylase Nocturnin

Abstract

Drosophila melanogaster curled, one of the first fly mutants described by T. H. Morgan >90 years ago, is the founding member of a series of curled wing phenotype mutants widely used as markers in fruit fly genetics. The expressivity of the wing phenotype is environmentally modulated, suggesting that the mutation affects the metabolic status of cells rather than a developmental control gene. However, the molecular identity of any of the curled wing marker mutant genes is still unknown. In a screen for starvation-responsive genes, we previously identified the single fly homolog of the vertebrate nocturnin genes, which encode cytoplasmic deadenylases that act in the post-transcriptional control of genes by poly(A) tail removal of target mRNAs prior to their degradation. Here we show that curled encodes Drosophila Nocturnin and that the gene is required at pupal stage for proper wing morphogenesis after eclosion of the fly. Despite the complex ontogenetic expression pattern of the gene, curled is not expressed in the developing wing, and wing-specific curled knockdown mediated by RNAi does not result in the curled wing phenotype, indicating a tissue-nonautonomous, systemic mode of curled gene function. Our study not only presents an entry point into the functional analysis of invertebrate nocturnins but also paves the way for the identification of the still elusive Nocturnin target mRNAs by genetic suppressor screens on the curled wing phenotype.

Figure 1.—

The Drosophila nocturnin (curled) gene: gene locus organization, deletion mutants, and phylogeny of Nocturnin proteins. (A) Organization of nocturnin (curled) gene locus with transcript isoforms no(cu)-RC, no(cu)-RD, and no(cu)-RE relative to the flanking genes CG18577 and SdhC at 86D7 on chromosome 3R. [Note: Coding parts of exons are marked by black, noncoding parts by white boxes; hatched boxes, Mg²⁺-dependent endonuclease-like domain (pfam03372); no(cu)-RC, -RD, -RE are the only no(cu) transcripts annotated in FlyBase r5.17.] Transposon integration line P{EP}no(cu)^GE22476 was used to generate nocturnin (curled) deletion mutants no¹ (cu³), no² (cu⁴), and no³ (cu⁵) as well as genetically matched no⁺ (cu⁺) control line no(cu)^GE22476rv. (B) Sequence alignment of vertebrate und invertebrate Nocturnin (NO) protein family members proves strong evolutionary conservation of amino acids involved in the catalytic function of yCCR4-related family proteins (amino acids identical to D. melanogaster NO are shaded in gray). Residues essential for the Mg²⁺-dependent endonuclease-like domain function are indicated as in Dupressoir et al. (2001): ▵, catalytic residue; □, residues involved in orientation and stabilization of catalytic residues; ○, for phosphate binding, and •, for Mg²⁺ binding residues; #, position of stop in no^stop12.2. The black bar illustrates the extent of the pfam03372 domain. (C) Phylogenetic tree analysis showing that Drosophila NO(CU) is the Nocturnin ortholog (Nocturnin subfamily shaded gray) among the four yCCR4-related proteins of the fly. (D) Northern blot analysis of adult flies shows nocturnin (curled) small S and large L transcript populations in no(cu)^GE22476rv controls and identifies no¹ (cu³) as transcript null mutation. No³ (cu⁵) specifically lacks no(cu) L transcripts, while no² (cu⁴) expresses a low abundance transcript slightly shorter than the no(cu) S transcript of control flies. SdhC transcript is unaffected in all no(cu) mutants. (E) Distally upward bent wing (left panel) and proximally crossed posterior scutellar bristle (right panel) phenotypes of no¹ (cu³) mutants compared to no(cu)^GE22476rv controls.

Figure 2.—

curled is nocturnin. (A) Schematic overview of molecular lesions in the cu¹ and cu² alleles at the nocturnin/curled gene locus. Boxes represent noncoding (open), coding (black), or miscoding (shaded) exons with the exception of the hatched box [cu(no) dsRNA] representing the cu(no) coding region targeted by a transgenic dsRNA snapback construct. Extent of genomic cu(no) rescue constructs without [cu(no)^+12.2] and with [cu(no)^stop12.2] stop mutation in a functional essential region of Nocturnin. Bars labeled with a, b, and c indicate position and extent of PCR amplicons used in B. (Note: Bar d, control amplicon from the brummer locus; M, DNA molecular size marker.) PCR-based genotyping in B confirms coverage of no¹ (cu³) by Df(3R)M86D and genomic integrity of SdhC in no¹ (cu³) mutants. No gross cu(no) locus aberrations in the cu² mutant allele. (C) cu(no) splice site mutation in cu¹ and frameshift mutation in cu². The no¹ (cu³) mutant wing phenotype is not complemented by cu² (D), and can be rescued by tissue-specific cu(no) cDNA (E), or cu(no)^+12.2 genomic (F) transgene expression. (G) cu(no) mutant phenocopy by ubiquitous expression of a cu(no) dsRNA transgene.

Figure 3.—

Complex and dynamic curled developmental gene expression and pupal function for wing morphogenesis. (A) Developmental Northern blot analysis detects curled S (±1.8 kb) and L (± 2.0 kb) transcript populations at all ontogenetic stages with the exception of early embryos, exclusively expressing maternally contributed cu S transcripts. (B and C) Tissue-specific expression of cu transcripts in embryos and third instar larvae. Expression in embryonic salivary glands (B) and in embryonic (B) and third instar larval (C) proventriculus and ring gland absent from cu³ deletion mutants. (D) Cytoplasmic intracellular CU-PC:EGFP localization upon targeted expression in third instar larval fat body. (Note exclusion from lipid droplets.) (E) Phenocritical period of curled wing morphogenesis function in pupae determined by in vivo RNAi. Percentage of curled winged phenotypes (black columns) and cu transcript expression levels (gray columns) scored in flies subjected to developmental time-controlled ubiquitous cu gene knockdown pulses during third instar larval and pupal development. Note: Arrows exemplify time points of heat-shock-mediated cu knockdown induction and error bars 95% confidence intervals. (F) Quantitative Northern blot analysis demonstrates starvation-responsive transcriptional upregulation of cu in adult male flies. sg, salivary gland; pv, proventriculus; rg, ring gland; st, starved.