Fly stocks and crosses
Batumi line laboratory stock was used in biochemical experiments and immunostaining. To generate recombinant chromosome 2 carrying the bic 1 lethal allele and FLAG.gNACβ transgene, the y1w*; Eney-whited700- pAc5.1.FLAG.gNAC-β.RetMI07200/CyRoi females were crossed to bic1L2/CyRoi males and the F1 Eney-whited700- pAc5.1.FLAG. gNAC-β.RetMI07200/ bic1L2 females were crossed to yw67c23, +/+ males. Male progeny of this cross with recombinant chromosome (marked whited700 and L2) Eney-whited700- pAc5.1.FLAG.gNAC-β.RetMI07200 bic1L2/+ were collected and individually crossed to yw67c23, +/CyRoi females. Males Eney-whited700- pAc5.1.FLAG.gNAC-β.RetMI07200 bic1L2/CyRoi were crossed to bic1L2/CyRoi females and the viable Eney-whited700- pAc5.1.FLAG.gNAC-β.RetMI07200 bic1L2/ bic1L2 individuals (lacking CyRoi and carrying the whited700 and L markers ) were traced/selected to evaluate the suppression effect of the bic1 lethality.
Germinal knockdowns
nos-Gal4 > UAS-gNACα-RNAi and nos-Gal4 > UAS-ubNACα-RNAi (VDRC) were performed separately or simultaneously using nos-Gal4 > UAS-gNACα-ubNACα-RNAi. The driver line was obtained from Bloomington Stock #25751, P{UAS-Dcr-2,D}1,w1118; P{GAL4-nos,NGT}40. RNAi lines include stocks from VDRC: # 102621 (gNACα (CG4415)), and # 36017 (ubiquitous NACα (CG8759)).
Constructs of plasmids
RT-PCR products of gNAC-β and gNAC-α ORFs were inserted into pAc5.1. FLAG or pAc5.1.HA carrying plasmids. Oligos for plasmid insertions:
pAc5.1.FLAG.gNAC-β (CG18313)
Xho1- 5'tataCTCGAGACAATGGATTTCAACAAGCGACAG
Apa1- 5' tataGGGCCCCTAATCTTCGTCCTCGGAGACCT ;
pAc5.1.HA.gNAC-α (CG4415):
Kpn1-5'tataGGTACCTTCCTCAAGATGGGTAAGAAGCAGA
Xho1- 5' tataCTCGAGGTTGTCGTTCTTCAGCAGCGC
Generation of transgenic lines expressing FLAG.gNACβ protein under рAc5.1 promoter
Transgenic strains carrying construct attBs-Eney-whited700- pAc5.1.FLAG.gNAC-β-attBsrev-pSK = aeca were generated by phiC31-mediated site-specific integration at the MiMIC site 48–50 in the Ret gene of chromosome 2 (Bloomington #43099, y1w*; MiRetMI07200/SM6a) and at the site (Bloomington #24862, yM{RFP[3xP3.PB] GFP[E.3xP3] = vas-int.Dm}ZH-2A w[*]; PBac{y[+]-attP-9A}VK00005) of chromosome 3. The vas-dPhiC31 strain bearing the phiC31 gene under the control of the vasa gene promoter on the X chromosome was used as an integrase source 49. The germ-line transformation of the embryos was performed according to standard protocol 51 with the approximately 40% efficiency of integration.
Generation of antibodies against germinal NACβ subunit
Here we generated rabbit anti His-tagged germinal NAC-β subunit antibodies using antigen sample as earlier described 10. Antibodies were purified by antigen affinity chromatography using Thermo Scientific AminoLink Plus Coupling Resin according to the manufacturer’s protocol (Thermo Fisher Scientific). The generated antiserum was shown to recognize exclusively the germ cells in testes and early embryos and was used in Western-blot analysis (dilution 1:1000).
Cell culture transfection, immunoprecipitation, and western-blot analysis
Transient transfections of S2 cells were performed with the help of FuGENE® HD Transfection Reagent (Promega# E2311) according to the manufacturer’s instructions. 3–4 days after transfection cells were harvested and subjected to immunostaining. For immunoprecipitation, anti-HA Magnetic Beads (#88836 Thermo Scientific) or anti-FLAG M2 Magnetic Beads (M8823 Sigma) were used. Protein samples (S2 cells or fly extracts) were applied to SDS-PAGE, transferred onto PVDF membrane according to standard protocols. The blots were analyzed using antibodies in a dilution 1:1000 against germinal NACβ, monoclonal mouse anti-FLAG M2 (Sigma F3165) and monoclonal mouse anti HA-Tag antibodies (mAB#2367 Cell Signalling Tech). Alkaline-phosphatase-conjugated anti-rabbit or anti-mouse antibodies (Sigma) were used as secondary reagent at a dilution of 1:20,000. Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories) in accordance with the recommendations of the manufacturer, signal was detected using the BioRad Chemi Doc MP Imaging System.
Ribosome’s isolation and 2D resolution of ribosomal proteins from testis
250 pairs of frozen testes were homogenized in a Dounce homogenizer in 1,5 ml of buffer containing 25mM Hepes [pH7.6], 100mM KCl, 5mM MgCl2, 1mM DTT, RNase inhibitor RiboLock (Thermo Scientific) at 40 units/ml, 0.015% digitonin, 1% NP-40, 0.5% sodium deoxycholate and 100 µg/ml cycloheximide. A protease inhibitor cocktail was used, as recommended by the company (Protease Inhibitor Cocktail Tablets, Roche). Cell fragments and mitochondria were removed by centrifugation at 12000g at 4оC for 20 min as described earlier [9]. 1,5 ml post-mitochondrial extract was centrifugated for 1 hour at 35,000 RPM (100000g) in a rotor Himac P50A3-0529, ultracentrifuge CP100-NX, at 4oC. 2D electrophoresis was performed at the «Human Proteome» Collective Use Center of the V.N. Orekhovich Federal State Budgetary Scientific Institution Research Institute of Biomedical Chemistry.
For first dimension, ribosome pellet or protein precipitate after dephosphorylation were suspended in 250 µl of buffer (7M urea, 2M thiourea, 4% [w/v] CHAPS, 1% [w/v] DTT, 2% immobilized pH gradient [IPG] buffer [pH 3–10], protease and phosphatase inhibitor cocktails [Roche Diagnostics, Mannheim, Germany]), then clarified for 10 min at 10000 g at 4°C. 120 µl of clarified protein solution mixed with 30 µl of rehydration buffer (7M urea, 2M thiourea, 2% CHAPS ,0.3% DTT, 0.5% IPG buffer [pH 3–11 NL], and 0.001% bromophenol blue) was used to prepare first-dimensional gel. 7-cm IPG gel strips (pH3-10) were rehydrated passively for 10 hour at 4°C. IEF was conducted at 20°C using Protean IEF Cell (“Bio-Rad”). Power supply was programmed in the gradient mode with voltages for four steps: first − 300V (00.30 min), second - gradient 1000V (00.30min), third – gradient 5000V (01.20min), fourth and hold – 5000V (00.25 min). Prior to the second dimension, the IPG gel strip was soaked for 10 min in equilibration solution 950 mM Tris-HCL [pH6.8], 6M urea, 2% SDS, 30% glycerol) containing 1% DTT. This process was followed by a 10-min incubation in the equilibration solution containing 5% iodacetamide. IPG gel strip was then placed on top of the second-dimensional stab gel and sealed using 1 ml of molten agarose with 0.5% TGS electrode buffer (24 mM Tris [pH8.3], 200 mM glycine, and 0,1% SDS). The second dimension SDS-PAGE was carried out using Hoefer miniVE vertical electrophoresis system [12% w/v gel concentration, 80 × 90 × 1 мм in size]. Precision Plus Protein Standards (BioRad) was used as a marker. Electrophoresis was performed at constant current (25 mA/gel) and 100 to 160 V for 1.5 h at room temperature. On completion of electrophoresis, the gel was rinsed in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS, and 20% methanol) and transferred to a PVDF membrane using a semidry transfer cell (Bio-Rad) following the manufacturer’s instructions.
Ribosome treatment by Calf Intestinal (CIP Sigma #4978) Alkaline Phosphatase
The treatment was performed according to following protocol: ribosomal pellet from 125 testes was suspended in 1xCIP buffer (0,1M NaCl, 0,05M Tris-HCl pH 7,9, 0,01M MgCl2, 1 mM DTT) ,1 x Tm Complete protease inhibitor mix (Roche), 20 units CIP, and reaction mix was incubated 120 min at 37oC. Dephosphorylation was completed by addition cold 10% trichloroacetic acid. Precipitate was washed twice by acetone before isoelectrofocusing.
Immunostaining
12–15-hour embryos were collected, dechorionated in bleach (2%) for 3 minutes and after thorough rinsing with water were devitеllinized in a 1:1 heptane/methanol mixture (-20°C) in a 1.5 ml microtube by gently shaking. Devitellinized embryos sinking in the methanol phase, were then rinsed 3 times with cold methanol. Embryos can be stored at -20°C until immunostaining. For immunostaining, embryos were gradually rehydrated with methanol-PBT (PBS with 0,1% Tween20), washed 3 times with PBTX (PBT with 0,3%Triton X100) and permeabilized in PBTX with 0,3% sodium deoxycholate (Sigma) for one hour. Then embryos were washed three times in PBTX and blocked with PBTX containing 5% normal goat serum (NGS, Invitrogen) for 1 hour. Embryos then were incubated first in specific primary antibodies in PBTX containing 3% NGS overnight at + 4°C and after washing 4 times in PBTX at room temperature, incubated the next overnight at + 4°C with secondary antibodies labeled with Alexa in a dark chamber. Embryos were mounted in Invitrogen SlowFade Gold Antifade reagent. The following primary antibodies were used: rabbit polyclonal anti-gNACβ (1:500), rat anti-VASA (1:200) (DSGB: AB_760351). Secondary antibodies were anti-rabbit IgG Alexa Fluor 546; anti-rat IgG Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific). Confocal microscopy was done using Zeiss LSM 900.
Testes and ovaries of adult (1–2 day old) males and females were dissected in phosphate-buffered saline (PBS) at 4°C, washed with PBT, fixed in 3.7% formaldehyde in PBT for 30 min at room temperature and then were processed and immunostained like embryos.
Quantification of NACs mRNA abundances
The abundances of testis-specific NACs were calculated by Salmon Galaxy ver. 1.5.1 using R6.22 transcripts fasta file and default settings. The source datasets and sequencing conditions are available in NCBI GEO, accession GSE101060.
Phylogeny of 69 Drosophila species
To estimate the phylogeny of Drosophila species, the 192 RefSeq and GenBank genomic assemblies of 70 species were downloaded from the NCBI. Each assembly was subjected to BUSCO v.4.1.4 27 analysis to identify the universal single-copy orthologs from OrthoDB (-l dipteria_odb10). The genomic assembly of Musca domestica (GCF_000371365.1_Musca_domestica-2.0.2) was also analyzed by BUSCO for further usage as the outgroup. 3,119 universal single-copy BUSCO orthologs that are present in at least 90% of 192 assemblies and 69 genomic assemblies (one assembly per species) having at least 90% of 3,119 single-copy orthologs were selected for the Drosophilidae phylogenetic analysis and the identification of NAC family proteins (Table S1). The concatenated multiple sequence alignments of the orthologous proteins (using MAFFT v7.47152 followed by alignment trimming with trimAl v.1.4 53 (-gt 0.5) resulted in 710,094 amino acid columns that were used to estimate the maximum likelihood species phylogeny using RAxML v.8.0 54 with the PROTGAMMAJTT model, rooted with the Musca domestica. We then used r8s 55 to estimate branch lengths in terms of millions of years with four calibration points 56 25–30 million of years (moy) for the common ancestor of D. pseudoobscura and D. melanogaster, 40 moy for the common ancestor of D. virilis and D. melanogaster, 6–15 moy for the common ancestor of D. yakuba and D. melanogaster, and 100 moy for the common ancestor of D. melanogaster and Musca domestica.
The identification of NAC family proteins
The initial identification of NAC genes in Drosophilidae genomic assemblies was performed with tblastn v. 2.6.0 57 (-evalue 10E-5) using NAC proteins from D. melanogaster as the queries. The hit regions extended with the additional 2000 bp on both sides were extracted from the genomes and were subjected to the determination of the open reading frames by AUGUSTUS v. 3.1.3 58. The identified open reading frames were confirmed as encoding NAC domains using InterProScan v.5.39 59 (IPR016641 for αNAC and IPR039370 for βNAC). If the analyzed genome was already annotated by the NCBI (40 genomic assemblies), then the accession number of the proteins was determined by blastp against the corresponding proteoms retrieved from the NCBI Protein database; otherwise (29 assemblies) the NAC protein was marked as ‘novel’. The multiple alignments of NAC proteins were carried out by the MAFFT v. 7.47152. The NAC domains of αNAC and βNAC were cut from the alignments and the positions including greater than > = 0.5 gaps were removed by trimAl, v.1.4 53. Phylogenetic analysis of NAC domains was performed using the FastTree program 60 with default parameters, with the WAG evolutionary model and the discrete gamma model with 20 rate categories. The tree structure was validated with bootstrap analysis (n = 100).