A nonsense mutation inBOD1 co-segregates with ID in a consanguineous Iranian family with four affected individuals

In a family with 4 female individuals with ID (Fig 1A) we performed multipoint linkage analysis based on the assumption of an autosomal recessive pattern of inheritance and a disease allele frequency of 0.001. We identified a single 4.3 Mbp interval on chromosome 5q (5q35.1–35.2) with a LOD score of 4.4 (S1 Fig) and sequenced the coding regions of all protein coding genes within the interval. This revealed a homozygous point mutation (NM_138369.2:c.334C>T; p.R112X) in the second exon of theBOD1 gene, which co-segregated with the disease (Fig 1A). The mutation was not found in 380 Iranian and 340 German control chromosomes and was absent in 200 Danish exomes [27]. In addition, theNM_138369.2:c.334C>T mutation was not found in the current data release (ESP6500SI-V2) of the Exome Variant Server (http://evs.gs.washington.edu/EVS/), NHLBI GO Exome Sequencing Project (ESP), Seattle, WA (accessed June 2015), containing exome sequencing results from 6503 individuals, nor in data from the 1000 Genomes Project [28], nor in the Exome sequencing Results from 60,706 unrelated individuals compiled by the Exome Aggregation Consortium (ExAC), Cambridge, MA (http://exac.broadinstitute.org, accessed February 2016). Moreover, our sequencing of controls and database search also revealed no other homozygous deleterious mutations in other parts of theBOD1 coding region.

The three affected females of the left branch of the family pedigree (V:2; V:3; V:6) suffered from moderate ID with an IQ of 50–55 (determined by Wechsler’s scale) in all three cases. In addition, these individuals presented with either primary (V:3) or secondary (V:2; V:6) amenorrhoea of unknown cause. Endocrinological tests and ultrasound investigations of the ovaries revealed no abnormalities. The affected individual in the right branch of the family pedigree (VI:1) presented with mild ID (IQ: 70–75). Brain MRI scans were performed on all four affected individuals, but revealed no consistent morphological abnormalities. All four individuals were obese or overweight but this phenotype did not co-segregate with the BOD1 mutation. From patient V:2 we obtained a lymphoblast cell line (LCL). In addition we were able to establish fibroblast cell lines from affected individuals V:2 and V:3. Cells derived from homozygous mutation carriers will be referred to asBOD1^-/- cells throughout the manuscript.

RT-PCR and sequencing analyses of control fibroblasts showed the presence of the full lengthBOD1 transcript (NM_138369.2), comprising all exons (1–4), represents the main isoform of the protein, which is 185 amino acids long and most likely its dominant functional form. In addition we detected three comparatively weakly expressed additional transcripts (Fig 1B and 1C), composed of the exons 1+3+4 (isoform b,NM_001159651.1, comprising 129 amino acids), 1+2+4 (ENST00000285908.5, encoding 129 amino acids) and 1+4 (ENST00000480951, encoding 85 amino acids). Quantitative PCR analyses of RNA preparations from control andBOD1^-/- cell lines further revealed thatBOD1 mRNA was absent in cell lines from both affected individuals (Fig 1D). This loss of BOD1 mRNA is likely caused by nonsense mediated decay (NMD) as mRNA levels of 3 of the 4 detected splice variants were increased to near-control levels following treatment with cycloheximide (Fig 1E). To confirm the loss of the main BOD1 isoform at the protein level, we investigated the patient cell lines by western blot, using a rabbit polyclonal antibody raised against recombinant full length GST-BOD1 [16]. In keeping with our quantitative PCR-results, this isoform was not detected in eitherBOD1^-/- cell line (Fig 1F).

BOD1^-/- fibroblasts progress through mitosis at an accelerated rate

We have previously reported that siRNA-mediated knockdown of BOD1 in HeLa cells produces profound chromosome biorientation defects and a block in mitotic segregation [16]. We therefore set out to determine whether abnormalities in cell cycle progression occur in cell lines derived fromBOD1^-/- individuals. We first examined cell cycle progression in WT andBOD1^-/- fibroblast cells (Fig 2A). This revealed a significant increase in the G1 population inBOD1^-/- cells compared to WT. Treating WT fibroblasts withBOD1 siRNA resulted in a similar accumulation of cells in G1 (Fig 1A and 1B) suggesting this change in cell cycle distribution is directly due to loss of BOD1. We observed no mitotic figures in WT fibroblast cells depleted of BOD1 by siRNA, suggesting that any cells with fully replicated DNA inFig 2A were in G2. In contrast, mitotic cells could be observed inBOD1^-/- cells suggesting these cells are capable of progressing through the cell cycle, but with a delay in progressing out of G1.

To detect any gross defects in mitotic chromosome segregation, we next examined the mitotic timing ofBOD1^-/-and control WT fibroblasts using DIC time-lapse microscopy, measuring the time from nuclear envelope breakdown (NEB) to anaphase onset (Fig 2C; seeMethods).BOD1^-/- fibroblasts progressed through mitosis rapidly, with 50% of cells completing mitosis in 20 min compared to 30 min for the control cells (Fig 2D). Similar results were obtained forBOD1^-/- LCL cells (S2A Fig).

Examination of fixedBOD1^-/- fibroblasts by immunofluorescence revealed no significant increase in cells with unaligned or malformed spindles (S2B Fig), suggesting only a subtle disturbance of mitotic regulation in these cells. This is surprising given the profound biorientation defects observed in BOD1-depleted HeLa cells. We therefore explored the properties of mitoticBOD1^-/- cells in more detail.

BOD1^-/- fibroblasts show mislocalisation of PLK1 and PP2A

Bod1 is required for proper chromosome alignment during mitosis and the proper phosphorylation of several substrates of the PLK1 and Aurora B (AURKB [MIM 604970]) protein kinases. This effect occurs through the modulation of PP2A-B56 activity [16]. To determine if these pathways were affected inBOD1^-/- cell lines, we investigated the localisation of PP2A-B56 and PLK1. In agreement with previously published observations in BOD1-depleted HeLa cells,BOD1^-/- fibroblasts had increased levels of PP2A-B56 at kinetochores (Fig 2E and 2F). Furthermore, PLK1 levels were reduced at the kinetochores ofBOD1^-/- cells compared to control fibroblasts (Fig 2G–2I), just as in Bod1-depleted HeLa cells. However, unlike BOD1 depleted HeLa cells,BOD1^-/- fibroblasts had increased PLK1 concentrations at centrosomes.

We also observed that total PLK1 protein levels were reduced in asynchronousBOD1^-/- fibroblasts (Fig 2J). However, when these cells were synchronised in mitosis using the Eg5 inhibitor Monastrol they exhibited PLK1 levels that were comparable to control fibroblasts (Fig 2K).

To determine if the changes in protein stability of PLK1 were limited toBOD1^-/- cell lines, we performed siRNA-mediated knock down of BOD1 in wild-type primary fibroblasts. The reduction in PLK1 levels was even more pronounced than that observed inBOD1^-/- cells with little or no detectable PLK1 protein (Fig 2L). PLK1 protein levels were partially recovered by co-depletion of BOD1 and PP2A-B56 (Fig 2L) suggesting that Bod1 inhibits PP2A mediated PLK1 destabilisation and that PP2A regulates PLK1 function throughout the cell cycle.

Since WT fibroblasts depleted of BOD1 do not enter mitosis, we next tested whether BOD1 has any role in mitosis in WT primary fibroblasts. We used immunofluorescence to localise endogenous Bod1 and observed prominent localisation to mitotic centrosomes and kinetochores (Fig 2M), suggesting that a mitotic function for Bod1 is present in these cells, but is masked in siRNA studies by an additional pathway in primary fibroblasts that results in G1 arrest. This G1 pathway is likely lost in highly transformed HeLa cells. To ensure the arrest was not limited to fibroblast cells, we depleted Bod1 from immortalised, non-transformed RPE1 cells and also observed a G1 arrest (S2C Fig). Once more, localisation of BOD1 to kinetochores and centrosomes was observed in untransfected RPE1 cells (S2D Fig), suggesting multiple roles for BOD1 throughout the cell cycle.

To check the specificity of the Bod1 siRNA, we rescued Bod1 depletion with plasmids expressing WT or constitutively active Bod1 [17] and observed rescue of PLK1 protein levels (Fig 2N) confirming the specificity of the effect on PLK1 by Bod1 siRNA. We conclude that BOD1 depletion in primary cells causes loss of PLK1 and that BOD1^-/- fibroblasts stabilise sufficient Plk1 to successfully progress through the cell cycle.

Changes in PLK1 function inBOD1^-/- cells

SinceBOD1^-/- cells are able to propagate and progress through the cell cycle, we hypothesised that they must have modulated the function of PLK1 to adapt to the presence of reduced PLK1 levels during interphase (Fig 2J). To test this hypothesis, we arrested cells at the G2/M boundary, where PLK1 is required for progression into M phase using RO3306, a CDK1 inhibitor. We then released cells into fresh medium containing increasing amounts of the specific PLK1 inhibitor BI2536. This assay uses the formation of bipolar mitotic spindles as a reporter of PLK1 function [17]. Control WT fibroblasts exposed to BI2536 showed a reduced number of bipolar metaphase spindles and an increased number of monopolar spindles as the concentration of BI2536 was increased (Fig 2O). By contrast,BOD1^-/- cells showed fewer monopolar spindles and only a small reduction in the frequency of bipolar spindles even at the highest concentrations of BI2536. This result suggests thatBOD1^-/- cells are hyposensitive to PLK1 inhibition relative to WT controls and that they have adapted to loss of BOD1 by either increasing or bypassing PLK1 function. Consistent with the former hypothesis,BOD1^-/- cells have increased levels of PLK1 at centrosomes (Fig 2G and 2I), a critical location for PLK1 function in centrosome and spindle maturation.

These results suggest that despite an overall reduction in PLK1 levels inBOD1^-/- cells, they have nonetheless adapted and progress through mitosis in a relatively normal manner. This conclusion is in line with our observation that homozygous mutation carriers do not show any gross developmental or structural brain abnormalities, as are often found in individuals affected by ID with mutations in proteins required for centrosome positioning and localisation (see e.g. Chavali et al. [29] or Kuijpers & Hoogenraad [30]). We therefore hypothesized that cognitive impairment caused by BOD1 deficiency might result from the disturbance of a different, possibly brain-specific functional aspect of BOD1.

BOD1 expression is maintained in postmitotic brain tissues

Quantitative PCR of RNA from different regions of normal human fetal and adult brain showed that all four BOD1 splice variants detected in lymphocytes and fibroblasts are present throughout the brain, at both investigated developmental stages (Fig 3A).

To address whether BOD1 might exert functions independent from cell-cycle regulation of PLK1, we compared BOD1 and PLK1 expression in different areas of the human brain by RNA-sequencing. In agreement with the low rates of dividing cells in these terminally differentiated tissues our results show that PLK1 expression drops to very low levels (Fig 3B) as compared to the expression rates observed in pluripotent cells. BOD1 expression, however, is maintained in most investigated brain tissues at about 20% of the expression levels observed in pluripotent cells, strongly suggesting that BOD1 plays a PLK1- and cell cycle-independent role in postmitotic brain cells.

BOD1 shows synaptic localization in mammalian neurons

Continuing on from the gene expression analysis we were interested in determining BOD1 localisation in mammalian neurons. We analysed murine corticoneuronal cells that were transfected with BOD1-GFP,at short incubation time (7h, to avoid overexpression artefacts). We observed a striking punctuate pattern with BOD1-GFP that co-localised with the synaptic marker Bassoon (Fig 4), which suggests a potential role for Bod1 in synaptic signalling.

BOD1 is required in postmitotic neurons for learning

To experimentally address whether BOD1 is required directly in neurons for cognitive processes, we took advantage of established animal model for ID disorders, the fruit flyDrosophila melanogaster. TheDrosophila genome encodes a single gene representing the human BOD1 protein family (BOD1, BOD1L1 and BOD1L2), termed CG5514. A multiple sequence alignment is shown as supplementalS3 Fig. From now on we refer to this so far uncharacterizedDrosophila gene as Bod1. We used the UAS-Gal4 system [31], the panneuronal promoter elav-Gal4 and three inducible RNAi lines carrying different constructs (Bod1^vdrc101981 Bod1^vdrc4542, and Bod1^HMS00720) to knockdown Bod1 specifically in postmitotic neurons. We subjected Bod1 panneuronal knockdown flies to a simple non-associative learning assay: the light-off jump reflex habituation paradigm. In this paradigm, which has previously uncovered learning defects in severalDrosophila models of Intellectual Disability [32–34] and in classic learning and memory mutants [35], flies are exposed to a repeated light off stimulus at 1 second intervals. Wildtype (wt) flies quickly adapt to the repeated stimulus and gradually suppress their initial jump response as a result of non-associative learning. In our experiments, flies of all tested genetic conditions showed high jump response towards the initial light-off stimulus, demonstrating that they properly perceived the stimulus and that their startle response was not compromised. We found that Bod1 knockdown flies failed to habituate to the presented light-off stimuli compared to their genetic background controls with normal Bod1 levels, and kept on jumping at high levels throughout the entire course of the experiments (Fig 5). This learning defect was consistent for two Vienna Drosophila Resource Center (VDRC) RNAi constructs, as well as for a non-overlapping independent short siRNA TriP construct (Fig 5A, 5B and 5C). To assess the significance of the habituation defects in both Bod1 knockdown models, flies were considered to have habituated once they failed to jump in five consecutive trials (no-jump criterion). Habituation was quantified as the number of trials required to reach the no-jump criterion (TTC, Trials to criterion). TTC of the Bod1 knockdown flies was 2.38–fold (Bod1^vdrc1105166, n = 143, p<0.001), 2.14-fold (Bod1^vdrc27445, n = 93, p<0.001) and 1.95-fold (Bod1^HMS00720, n = 70, p<0,001) increased over their respective controls (Fig 5A’, 5B’ and 5C), revealing significant habituation defects in all three Bod1Drosophila models.

The efficacy of the utilised RNAi constructs was confirmed by qPCR on RNA isolated fromDrosophila 3^rd instar larvae upon ubiquitously-induced knockdown. As Bod1^vdrc27445 and Bod1^vdrc105166 contain almost identical siRNA hairpins, only Bod1^vdrc105166-mediated knockdown was assessed and revealed 23% remaining gene expression; p = 0.0012, student’s t-test; supplementalS1 Table.Bod1^HMS00720-mediated RNAi reduced levels ofBod1 to a lesser but still very significant extend, resulting in 37% remaining gene expression (p = 0.0086, student’s t-test;S1 Table).

We conclude that conditional knockdown of Bod1, induced specifically in neurons, does not affect the overall startle response/acute fitness of the flies but leads to specific and highly consistent learning defects.

Synapse biology is crucial for learning and has been proposed to play a central role in ID [36–38]. Therefore, and because of the observed Bod1 localisation to synapses in cultured neurons, we investigated the consequences of Bod1 knockdown on synapse development. TheDrosophila larval Neuromuscular junction (NMJ) was investigated in elav-Gal4 induced panneuronal Bod1 knockdown larvae using an antibody against dlg1 that visualizes the overall morphology of synaptic terminals. Knockdown of Bod1 with both Bod1^vdrc27445 and Bod1^vdrc105166 lines induces modest but highly significant abnormalities in synapse branching (Fig 5D)The average number of branching points was 2.7 versus 1.6 in the appropriate genetic background control (Bod1^vdrc105166 versus control); p<0.00001) and 2.5 versus 1.3 (Bod1^vdrc27445 versus control; p<0.005). Other synaptic parameters such as length or perimeter of the synaptic terminal were not consistently changed. There was no significant NMJ defect in the weaker Bod1^HMS00720 knockdown condition (1.5 in Bod1^HMS00720 versus 1.4 in control). We conclude that Bod1 is required for habituation and may control synaptic branching.

Ethics statement

The ID study was performed in agreement with the approval (2100) of the ethics committee of the Charité University Medicine Berlin ("Ethikkommission der Charité—Universitätsmedizin Berlin"), Germany. Written consent for the intellectually disabled individuals was provided by their parents.

Sample collection, DNA and cell line preparation

The pedigree of the family reported here is shown inFig 1A. Blood samples for DNA preparation were collected from the parents and all children of both branches and genomic DNA was extracted using a standard method. For the index patient (V:2), Fragile X was excluded by PCR and Southern blot analysis. Filter-dried blood of the index patient was screened by tandem mass spectrometry to exclude disorders of the amino acid, fatty acid (e.g. phenylketonuria) or organic acid metabolism [61,62]. Standard 450 G-band karyotyping was performed in order to exclude cytogenetically visible chromosomal aberrations.

An additional blood sample from patient V:2 was used to establish an EBV transformed lymphoblastoid cell line and skin biopsies were taken from individuals V:2 and V:3 to isolate and culture fibroblast cells.

Whole genome linkage analysis and mutation screening

Genotyping was performed with Human Mapping 10K Array Version 2 (Affymetrix). Multipoint parametric linkage analysis was performed using Allegro [63] applying an autosomal recessive pattern of inheritance and disease allele frequency of 0.001. Sanger sequencing was performed and the whole coding and exon-flanking regions in the interval were screened (The primer sequences are available upon request). The resulting sequences were analyzed using the CodonCode aligner software.

mRNA assays

Total RNA from the fibroblast and EBV transformed lymphoblast cultures was isolated by using TRIzol reagent RNA extraction protocol (Invitrogen). In addition, commercially available RNA (BioCat) was used from various tissues as indicated inFig 3.

Semiquantitative RT-PCR was performed in two steps, after Dnase treatment of the RNA with RQ1 RNase-Free DNase (Promega), cDNA was synthesized with SuperScriptIII reverse transcriptase kit (Invitrogen) together with random hexamers and followed by Polymerase Chain Reaction for 35 cycles with primers in both sides of the gene (Forward primer:CATCGTGGAGCAGCTCAAG and Reverse primer:GCACTCTTATGTAACCGAATC) to amplify all the possible splice variants. Subsequently each of the splice variants were verified by isoform specific RT-PCR (The primer sequences are available upon request) as well as Sanger sequencing.

Isoform specific quantitative PCR was performed in 20 μl volumes and carried out in the ABI PRISM 7900 HT Sequence Detection System using a 96-well format. Cycling parameters: 10 min at 95oC followed by 40 cycles of 15s 95oC and 1 min 55oC. Amplification plot and predicted threshold cycle (Ct) values were obtained with the sequence Detection Software (SDS 2.1, PE Applied Biosystems).

We used absolute quantification to quantify unknown samples by interpolating their quantity from a standard curve. To construct the standard curve four-fold dilutions (1, 0.5, 0.25, 0.125) of a total RNA preparation from control cDNA were used. Negative controls (“no template control”, NTC) were used to verify amplification quality and to exclude contamination or primer-dimer artifacts. The gene used for normalising expression wasGAPDH.

NMD analysis

The fibroblast cells from two individuals affected by ID (V:2 and V:3) and two controls were cultured into two sister flasks, one treated with cycloheximide (CHX) (Sigma) at the concentration of 500 μg/ml and the other one non-treated (just added DMSO). After 7.5 hrs of incubation in 37C / 5%CO2, the cells were harvested, RNA was extracted and isoform specific quantitative PCRs were carried out.

Cell lines and cell culture

HeLa S3 cells were maintained in EMEM (Lonza), Primary Fibroblasts were maintained in Quantum 333 media (PAA) or FibroPlus 333 (Capricorn-Scientific) and EBV-transformed lymphoblasts were maintained in RPMI 1640 media (Gibco). All media was supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin. Quantum 333 and FibroPlus 333 media was also supplemented with 10 ng/ml bFGF (Cell Signalling Technology). Cell lines were maintained at 37°C with 5% CO₂ in a humidified incubator.

Fibroblast cell lines were electroporated using the Neon Transfection system (Life Technologies) as per the manufacturer’s instructions using a single pulse of 20 ms and 1600 V. Medium GC content control siRNA and BOD1 Stealth siRNA (5’-GCCACAAAUAGAACGAGCAAUUCAU-3’) were supplied by Life Technologies. The specificity of these reagents and absence of off-target effects were reported previously [16,17]. Briefly, BOD1 Stealth siRNA does not target Bod1L or Bod1L2 mRNA and all phenotypes associated with the BOD1 Stealth siRNA can be rescued using exogenous siRNA-resistant BOD1 expression constructs.

Unless otherwise indicated RO3306 was used at 9 μM, Monastrol at 100 nM and BI 2536 at 12.5 to 200 nM.

Human fibroblast immunostainings and microscopy

Time-lapse imaging and fluorescence microscopy was performed as described [16]. Non-adherent lymphoblast cells were gently centrifuged onto the surface of a Labtek imaging chamber and constricted close to the coverslip surface by the addition of 50% Matrigel (BD Biosciences). During DIC timelapse imaging of lymphoblast and fibroblast cell lines 10 z sections, 3 μm apart were taken every 4 min for 18 hr on a DeltaVision Spectris (Applied Precision) fitted with an environment chamber (Solent) maintained at 37°C. All images were stored and manipulated using OMERO [64] or Photoshop (Adobe). Images were quantified using OMERO and m-tools as described [17]. Statistical significance was determined using Mann-Whitney Rank Sum Tests. Nuclear envelope breakdown (NEB) was defined as the time when a clear delineation of the nuclear envelope was no longer visible and the volume occupied by chromosomes began to expand. While not an exact measure of NEB, this approach combined with the 4 min interval between images provided sufficient accuracy to reveal differences in mitotic timing in these cell lines.

Rabbit anti-BOD1 [16] and mouse anti-B56α (BD Biosciences) were used at 1:100. Mouse anti-PLK1 (Upstate), and alpha-tubulin (Sigma) were used at 1:500. Human ACA (CREST) autoantisera (a kind gift from Sara Marshall, Ninewells Hospital, Dundee), were used at 1:1000. All fluorescently labelled secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.

RNA-sequencing

RNA Expression profiling was carried out on a SOLiD5500XL Sequencing platform as previously described [65], using RNA preparations form human embryonic stem cells (hES), induced pluripotent stem cells (iPSC) and commercially available RNA (BioCat) from various adult brain tissues as indicated inFig 3.

Isolation of murine primary corticoneuronal cells

The cortex was extracted from mouse embryonic brain at an age of E14.5. One day prior to sacrificing the mother plates were prepared as follows: Sterilized cover slips were placed tissue culture wells (12-well plates). The wells were then coated by incubating them for 30 min at room temperature or over night at 4°C with poly-D-Lysine/Laminin (poly-D-lysine 1:500 and laminin 1:50 in PBS, 1 ml per well). The wells were then washed twice with 1 ml PBS before adding 2 ml neurobasal medium to each well and then incubating at 37°C (8% CO₂).

The pregnant mother was sacrificed by neck fracture, then the embryos were removed from the womb and placed in a sterile Petri dish containing PBS.

The preparation of corticoneuronal cells was carried out on a sterile flow bench. Embryos were briefly washed with PBS and then transferred in a fresh dry Petridish. Next the heads were removed and the brains excised. The brains were suspended in 1–2 ml DMEM, each. The Brain stem and brain lobes were separated under a stereoscope and the cortex removed, taking care to separate the cortex from the hippocampus. Cortex halves were then transferred in a chilled DMEM-Tube on ice. Next, single cells from isolated embryonic cortices were prepared: 5 ml trypsin was added and incubated for 7 min at 37°C (5%CO₂), swirling once in between. Trypsin activity was then stopped by adding DMEM + 10% FCS and centrifugation (200xg for 3 min). This step was repeated twice. Finally the DMEM was carefully removed and 2 ml neurobasal medium were added, before decollating the cells using a narrowed pasteur pipette. Cells were then diluted by adding 28 ml neurobasal medium, counted and seeded to a density of 1.5x105 cells per cm² in the prepared 12 well plates. After approximately 1 hr of incubation at 37°C (8% CO₂) the medium was changed in order to remove unwanted cells. The next change of medium was after day 3–4. Subsequently the medium was changed every 7^th day. On the 7^th day after seeding (or later) cells were used for experimental applications.

Transient transfection of murine primary cortical neurons

Cells were transfected by pEGFP-BOD1 (wt) or pEGFP-N1 vector (Control) at days 7 and 9 after preparation. A solution-A (containing 0.1 μg of plasmid DNA and 100 μl OptiMEM), and a solution-B (with 2 μl lipofectamine and 100 μl OptiMEM) were prepared and kept for 5 min at room temperature. Subsequently, solutions were mixed and incubated at room temperature for 20 min. Meanwhile, cell culture medium was replaced by transfection medium (antibiotic free culture medium). The transfection mixture was slowly added to the cells which then were incubated at 37°C (8% CO₂). On both days cells were fixed with PFA 4% at two time points (6 and 8 hr).

Fly stocks

Fly stocks were kept on standardDrosophila diet (cornmeal/sugar/yeast) at 25°C, in a12h:12h light/dark cycle. For the habituation experiments, flies were reared and tested at 25°C and 70% humidity, 28°C and 60% humidity was used during synapse morphology experiments and to generate ubiquitous RNAi-mediated knockdown for quantitative PCR (qPCR). Inducible RNAi lines against theBOD1 Drosophila ortholog CG5514 (vdrc105166, vdrc27445) and their corresponding genetic background control lines (vdrc60100, vdrc60000) were obtained from ViennaDrosophila RNAi Center [66]. A nonsense RNAi line, targeting aC.elegans-specific gene, was obtained from K. Keleman. Inducible short-hairpin RNAi line HMS00720 (BL32928) and its corresponding genetic background control liney v; attP2,y+ (BL36303), generated by Transgenic RNAi Project (TRiP), were obtained from Bloomington Drosophila Stock Center. The ubiquitous actin-Gal4 driverw¹¹¹⁸; P(w[+mC] = Act5c-Gal4)/CyO, also obtained from BloomingtonDrosophila Stock Center, was used to generate RNAi-mediated knockdown for qPCR. In-house assembled panneuronal elav-Gal4 driver lines were used for habituation (2xGMR-wIR; elav-Gal4, UAS-Dicer2)) and synapse morphology experiments (UAS-Dicer2; elav-Gal4). In all experiments, progeny of a cross between these Gal4-driver lines and the genetic background of the respective RNAi line was used as a control.

Light-off jump habituation

The light-off jump habituation assay was performed as previously described [32] with minor adaptations. Briefly, 3–7 day old individual male flies (Bod1^vdrc105166, Bod1^HMS00720) or female virgin flies (Bod1^vdrc24445) were tested for jump responses in two independent 16-unit light-off jump habituation systems. 32 flies (16-flies/system) were simultaneously exposed to series of 100 short (15ms) light-off pulses with a 1s interval between the pulses. The noise amplitude of wing vibration following every jump response was recorded for 500ms after the start of pulse and a carefully determined threshold was applied to annotate jump responses. Data were collected and analyzed by custom-made Labview Software (National Instruments). High initial jumping response to light-off pulse decreased with growing number of pulses and flies were considered habituated when they failed to jump in 5 consecutive trials (non-jump criterion). Habituation was scored as the number of trials required to reach the non-jump criterion (Trials To Criterion, TTC). Main effects of genotype (mutant vs control), day and system on log transformed TTC values were tested using linear model regression analysis (lm) in R statistical software (R version 3.0.0 (2013-04-03)). A nonsense RNAi line targeting a C.elegans-specific gene (KK library construct) was also tested and did not affect habituation.

Analysis ofCG5514 mRNA levels from wholeDrosophila larvae by qPCR

Total RNA from 3rd instar larvae (3 biological replicates) was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen). During the isolation procedure, samples were treated with RNase-free DNase Set (Qiagen). RNA isolated from Bod1^HMS00720 and its respective genetic background control was used directly for cDNA synthesis. RNA isolated from Bod1^vdrc105166 and its respective genetic background control was further processed by Oligotex mRNA Mini Kit and purified PolyA+ RNA was used for cDNA synthesis. First strand cDNA synthesis was performed using iScript cDNA Synthesis Kit (Biorad). Gene expression was analysed by real-time PCR (7900HT Fast Real-Time PCR system, Applied Biosystems). PCR reactions were performed in a volume of 25μl containing 150 nM primers and GoTag Green Mastermix (Promega). Primer sequences used for amplification ofCG5514: 5’-ACAACAGTGGGGAGCCAG-3’ and 5’- CCTGTGCTAGTCGTCTCCG-3’. The amplicon spans the HMS00720 cleave site and should effectively detect the initial cleavage step of RNA interference. To determine the efficiency of the initial cleavage step of long vdrc105166 siRNA hairpin, the primers were designed to detect the region within the 5’ cleavage fragment, and qPCR was perfomed on PolyA+ mRNA. This ensures that in case of successful initial cleavage, regions 5’ to the cleavage site would not be amplified in qPCR reaction[67]. Pol II was used as reference gene, primer sequences: 5’- TCAGAGTCCGCGTAACACC-3’, 5’- TGGTCACAAGTGGCTTCATC-3’.

Analysis of synaptic morphology

Wandering L3 larvae were dissected and fixed in 3.7% paraformaldehyde for 30 minutes. Males were selected for Bod1^vdrc105166 and female larvae for the X-linked Bod1^vdrc24445. Type 1b neuromuscular junctions (NMJs) at muscle 4 were visualized using the primary antibody anti-dlg1 (1:25, Developmental Studies Hybridoma Bank) in combination with the Zenon Alexa Fluor 568 Mouse IgG1 labelling kit (Invitrogen). NMJ images were acquired using a Leica automated high-content microscope. Individual synapses of segments A2 –A5 were imaged and quantified using an in-house developed Fiji-compatible macro. Synaptic parameters were compared between the RNAi line and the corresponding genetic background control line using a student’s T-test.

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Supplementary Materials

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.