Drosophila melanogaster MLE Helicase functions beyond dosage compensation: molecular nature and pleiotropic effect of mle[9]

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Abstract

MLE of D. melanogaster is a conserved protein in higher eukaryotes, an ortholog of human DHX9 helicase. In mammals, this helicase has been shown to participate in different stages of gene expression. In D. melanogaster, the role of MLE as one of the components of the species-specific Dosage Compensation Complex has been extensively studied. However, the role of MLE in other processes has remained poorly understood. In this work, for the first time, the mle[9] mutation is mapped at the molecular level and shown to be caused by a deletion resulting in the loss of a highly conserved motif III in the catalytic core of the molecule. Thus, mle[9] specifically disrupts the helicase activity of the protein without affecting the function of other domains. The study of phenotypic manifestations of the mutation in females showed that in the homozygous state it has a pleiotropic effect. Without affecting survival, it significantly reduces fertility and lifespan. In addition, the duplication of scutellar macrochaetae was observed with high frequency. These results confirm that in D. melanogaster MLE helicase is involved in a wide range of gene expression regulation processes distinct from its role in dosage compensation.

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About the authors

G. A. Ashniev

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: julia.v.nikolenko@gmail.com
Russian Federation, Moscow, 119991

S. G. Georgieva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: julia.v.nikolenko@gmail.com
Russian Federation, Moscow, 119991

J. V. Nikolenko

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: julia.v.nikolenko@gmail.com
Russian Federation, Moscow, 119991

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2. Fig. 1. Molecular mapping of the mle mutation[9] at the DNA and mRNA levels. a is a schematic representation of the mle gene and its transcripts. mle-RA is a transcript encoding a full-length protein; mle–RC is a hypothetical transcript. Asterisks indicate the sites of HincII restriction endonuclease; arrows indicate the position of probes 1, 2 and 3 used to map the deletion; a black triangle indicates the exact position of the deletion that we have established. The black segments indicate the position of the probes for measuring the level of mle transcription “above” (4) and “below” (5) deletion; b is a fragment of the nucleotide sequence in the area of mle deletion[9] in DNA and cDNA. In DNA, the intron sequence is indicated in lowercase letters, the exon sequence is indicated in uppercase letters; the sequence deleted in the mle mutation[9] is highlighted in gray, AG dinucleotides are emphasized – acceptor splicing sites in the wild–type gene and in the mutation.

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3. Fig. 2. The structure of the MLE protein in mle mutants[9]. a is the amino acid sequence of the MLE protein. The deletion is highlighted in gray; b is the MLE domain structure (cit. according to [7] with changes). The areas corresponding to certain domains are indicated in gray, and the linker areas are indicated in white. Two dsRNA binding domains are presented – dsRBD1 and dsRBD2, the minimal MTAD transactivation domain, the catalytic “core” consisting of the recA1 and RecA2 domains and the helicase-associated HA2 domain, the binding region of the oligosaccharide/oligonucleotide OB-fold, the nuclear localization signal NLS, the glycine-rich C-terminal domain Gly. Conservative motifs are indicated by Roman numerals, the position of the deletion is indicated by a black arrow; c is the alignment of the sequence containing motif III in representatives of different species. Similar in different types of a.o. are highlighted in bold, identical to a.o. highlighted in bold and underlined. The deletion of mle[9] is highlighted in gray.

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4. 3. The mle mutation[9] does not disrupt gene expression in vivo in homozygous females. a – transcription of mle in females with mle mutation[9]. The mle transcription level in imago was measured relative to the transcription level of the control gene β-Tubulin56D. Probes located “above” (5ʹ) and “below” (3ʹ) the mle deletion were used[9]. Heterozygous females of the same line (+/–) were used as a positive control. Error bars indicate the standard measurement error; b – Western analysis of MLE protein expression in larvae and pupae. L – wandering faces L3, P – pupae, age one day; “–/–” – homozygous, “+/–” – heterozygous, “+/+” – Oregon R. A nuclear extract of 10 individuals is applied to each track of the gel.

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5. Fig. 4. Mutation of mle[9] in the homozygous state reduces the lifespan of females. a – survival curves at a temperature of 24 °C. mle[9] – homozygous females, mle[9]/CyO and mle[9]/+ – heterozygous females with curved and straight wings, respectively, +/+ – females of the Oregon R line; b – average life expectancy. The error bars indicate the standard error of the mean, * – p < 0.001.

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6. Fig. 5. The mle mutation[9] in the homozygous state reduces the fertility of females. a is the dynamics of female fertility during the first three weeks of life. mle[9] – homozygous females, mle[9]/CyO – heterozygous females, +/+ – females of the Oregon R line. The error bars indicate the standard error of the average; b – the average number of offspring for the first 22 days of life, * – p < 0.001.

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7. 6. The mle mutation[9] affects the formation of scutellar macrochetes (asc). a – doubling of the anterior scutellar macrochete on one (left) or both (right) sides of the breast in homozygous females. The white arrow indicates the normal location of macrochetes, the black arrows indicate the mutant phenotype; b – the frequency of occurrence of the mutant phenotype in homo– (mle[9]) and heterozygous (mle[9]/CyO) females; 4*asc - females with a doubling of macrochetes on both sides, * p < 0.001.

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