Dis3L2 null mutants show widespread tissue overgrowth

Our previous work, using RNA interference, demonstrated that Drosophila are an excellent model organism for the study of how Dis3L2 functions to control cell proliferation and tissue growth [1]. Since RNA interference has its limitations due to residual activity, we used CRISPR-Cas9 to generate a dis3L2 null mutant as an optimal genetic model to understand the molecular pathways regulated by Dis3L2 to control proliferation. Using this approach, we produced a dis3L2 mutant line with an 8bp deletion (dis3L2¹²) in which no Dis3L2 protein is produced (Figs 1A, 1B and S1A). In parallel, we also generated an isogenic control line which remained unedited or was repaired correctly (dis3L2^wt (Fig 1A and 1B)). Crucially, dis3L2¹² flies showed wing overgrowth similar to that previously observed in our RNAi flies (Fig 1C). To genetically confirm the null nature of the allele we created hemizygous flies by crossing the mutant dis3L2¹² chromosome over the smallest available deletion that includes the dis3L2 locus (DF(3L)Exel6084). Whilst hemizygous flies containing a single wild-type copy of Dis3L2 (dis3L2^wt/Df) showed no phenotype those containing the mutant chromosome (dis3L2¹²/Df) showed significant wing overgrowth (13%) that was not different to homozygous dis3L2¹² flies (Fig 1C). As with our previously published data the wing overgrowth is primarily driven by an increase in cell number in addition to a small (2%) increase in cell size (Fig 1D and 1D’).

Fig 1

dis3L2¹² mutants show overgrowth phenotypes.

A) Schematic showing location and details of the dis3L2¹² mutation. Coding exons are marked in orange and 5’ and 3’ UTRs in blue. RC and RA denote the two dis3L2 isoforms and red letters denote the start codon. B) The 8bp mutation in dis3L2¹² flies results in a complete absence of the Dis3L2 protein. C) dis3L2¹² wings (red) are significantly larger (14%) than control dis3L2^wt wings (blue). Hemizygous dis3L2¹²/Df wings are not significantly different from dis3L2¹² wings demonstrating that it is a null mutation. n = 16–19, error bars represent 95% CI, **** = p<0.0001, ns = p>0.05, scale bar = 100μm. D/D’) The increased wing area is mainly driven by an increase (12%) in cell number (D) and a small (2%) increase in cell size (D’). n = 13, error bars represent 95% CI, * = p<0.05, **** = p<0.0001. Representative images of one of 5 wing locations used to calculate wing area is shown for dis3L2^wt and dis3L2¹² wings. Each cell is marked by a trichome (hair) in the upper and lower cell layer. E) dis3L2¹² wing, haltere and leg imaginal discs are significantly larger (25%, 30%, 36% respectively) than their control counterparts. n = 8–33, error bars represent 95% CI, ** = p<0.01. F) Consistent with an increase in wing area, PH3 staining of wing, haltere and leg imaginal discs demonstrates an increased number of proliferating cells in dis3L2¹² tissues. n = 5–16, error bars represent 95% CI, scale bar = 50μm. G) dis3L2¹² mutants are significantly larger than dis3L2^wt flies. Calculated using measurements of various regions of the adult fly (A-G, S7 Fig) and total mass. n = 15–21, error bars represent 95% CI, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. H) SUnSET labelling performed on wing imaginal disc samples shows increased global translation in dis3L2¹² discs compared to dis3L2^wt. Representative lanes from the same blot are shown along with Tubulin loading control used for quantification. Puro–represents a disc sample incubated in media only without Puromycin. n = 8–14, error bars represent 95% CI, ** = p = 0.0057.

To determine the tissue specificity of dis3L2 mutations, we checked for overgrowth of other tissues in our null mutants. These experiments showed that the overgrowth is not restricted to the wing but is also observed in the haltere and leg imaginal discs (Fig 1E). Phosphohistone H3 staining confirmed the increase in dis3L2¹² tissue size is driven by an increase in proliferation in wing, haltere and leg imaginal discs (Fig 1F). Finally, although we observe a general increase in adult fly size (Fig 1G) we do not see a similar increase in size of dis3L2¹² L3 larvae, or larval salivary glands suggesting the overgrowth effects are restricted to imaginal tissues (S1B Fig).

Increased growth would be expected to require increased protein translation. To assess the changes in global translation within the wing imaginal disc we used surface sensing of translation (SUnSET) labelling. SUnSET labelling uses puromycin as an analogue of tyrosyl-tRNA and therefore when cells are incubated with low amounts of puromycin it is added into the elongating peptide chain. The amount of incorporated puromycin over a given time is detected with a monoclonal antibody which allows the measurement of nascent translation. Using this approach, we confirmed an increase in translation within the wing imaginal discs (Fig 1H).

In addition to the overgrowth phenotype we were also able to confirm the male fertility phenotype observed by Lin and colleagues (S1C Fig) [11]. dis3L2¹² male flies were completely infertile with seminal vesicles devoid of sperm. We further confirmed the specificity of the infertility phenotype in male flies where dis3L2 had been ubiquitously knocked down using Tubulin-GAL4 and UAS-dis3L2^RNAi (S1C Fig). Finally, both male and female dis3L2¹² show a decreased lifespan compared to dis3L2^wt flies (S1D Fig).

Dis3L2 catalytic activity is required to maintain control of tissue growth

To confirm that loss of Dis3L2 is indeed driving the overgrowth phenotype we generated a number of transgenic lines to allow specific genetic rescue. There are two annotated isoforms of Dis3L2 which differ through the use of an alternative start codon resulting in two protein products of 1032 amino acids (isoform PA) and 1044 amino acids (isoform PC). To ensure that re-expression of Dis3L2 was to a level comparable to wild-type we used the site-specific recombination attB/attP system to allow the specific insertion of Dis3L2 cDNA into a genetic region which would result in mild expression. This mild expression was confirmed by Western blotting where driving ubiquitous ectopic expression of these lines using actin5C-GAL4 resulted in a mild ~2 fold overexpression (Figs 2A and S2A) in dis3L2^wt flies. Re-expression of Dis3L2 protein throughout dis3L2¹² mutant flies was achieved to 143% and 170% the level of control flies for PA and PC respectively (Compare -/PA and -/PC to +/- in Fig 2A and see S2A Fig for quantification). Expression of both Dis3L2 isoforms (PC) or only PA specifically in the cells fated to form the wing (using nub-GAL4) results in a complete rescue of wing area to that of dis3L2^wt wings whilst the fly mass remained significantly larger (Figs 2B and S2B). To confirm the specificity of the phenotypic rescue we extended these findings using the engrailed-GAL4 (en-GAL4) driver to allow specific re-expression of Dis3L2 in the posterior compartment of the wing whilst the anterior area remained mutant for Dis3L2, therefore acting as an internal control. This revealed a specific rescue of the posterior area in en-GAL4 driven rescue wings whilst the anterior area remaining significantly larger (S2C Fig). Importantly, driving mild overexpression of Dis3L2 with these UAS lines with either nub-GAL4 or en-GAL4 has little effect on wing area alone (S2D and S2E Fig). These phenotypic rescues show that the loss of Dis3L2 is indeed responsible for the overgrowth phenotype and that isoform PA is the major isoform in Drosophila to regulate tissue growth.

Fig 2

Ectopic expression of catalytically active dis3L2 rescues the wing overgrowth phenotype.

A) UAS-dis3L2^PA, UAS-dis3L2^PC, or UAS-dis3L2^ND can be used to express Dis3L2 in dis3L2¹² animals. UAS constructs were driven in either dis3L2^wt (+) or dis3L2¹² (-) animals with the ubiquitous driver act-GAL4 with Western blots performed on whole female flies. See S2A Fig for quantification. B) Expression of both Dis3L2 isoforms (PC) or only the shorter isoform (PA) in the wing pouch of the wing imaginal disc is sufficient to completely rescue wing area in dis3L2¹² mutants. Expression of a catalytically dead Dis3L2 (ND) does not rescue the phenotype, whilst expression of human DIS3L2 (hDIS3L2) also shows no significant rescue at 25°C. n = 14–65, error bars represent 95% CI, statistics shown refer to a comparison between the test genotype and control **** = p<0.0001, *** = p<0.001, ns = p>0.05. C) When driven at 29°C the human DIS3L2 construct (hDIS3L2) shows a partial (~50%) rescue of wing area. n = 16–28, error bars represent 95% CI, **** = p<0.0001, *** = p<0.001.

Next, we asked if the catalytic activity of Dis3L2 was required to control proliferation by creating a nuclease dead transgenic line (UAS-dis3L2^ND) carrying a single amino acid substitution (D580N) which has previously been shown to abolish Dis3L2 catalytic activity [13]. To create this line, we used the same targeted insertion site which resulted in expression comparable to the wild-type constructs (Figs 2A and S2A). However, unlike the previous rescue experiments, specific expression of dis3L2^ND results in no change in dis3L2¹² wing area when driven by either nub-GAL4 (Fig 2B) or en-GAL4 (S2C Fig) demonstrating the catalytic activity of Dis3L2 is indeed essential to control proliferation in the wing imaginal disc. Finally, to assess functional homology between the Drosophila and human Dis3L2 proteins we also created a line to allow us to express human DIS3L2 (Ensembl isoform 202, 885aa) in our dis3L2¹² flies (S2F Fig). Whilst expression of human DIS3L2 in a dis3L2¹² mutant background did not rescue wing overgrowth at 25°C (Fig 2B), increasing the temperature to 29°C resulted in a 50% rescue of wing area towards that of dis3L2^wt flies also reared at 29°C (Fig 2C). The requirement of an increased temperature may be due to the fact that the human protein would function optimally at 37°C. The difference in rescue may also be due to the increased efficiency of the GAL4 system at a higher temperature and therefore more human protein would be produced. These data therefore demonstrate the catalytic activity of Dis3L2 is required to control proliferation and tissue growth and that there is at least partial functional homology between the human and Drosophila Dis3L2 proteins.

Overexpression of Dis3L2 results in reduced proliferation

In addition to assessing the phenotypes consequent upon the loss of Dis3L2 we also investigated the in-vivo effect of strong overexpression of Dis3L2 using the wing imaginal disc as a model tissue. To achieve this, we used a publicly available line containing a P-element insertion, with an upstream activating sequence (UAS) 8bp upstream of Dis3L2 to achieve strong overexpression of the DIS3L2 protein (Fig 3A). Overexpression of Dis3L2 in the posterior compartment of the wing imaginal discs (en-GAL4) resulted in a significant and specific reduction in the area of posterior compartment of both the wing and the wing imaginal disc (20% and 13% respectively) (Fig 3B and 3C). In these experiments the anterior area, which functions as an ideal internal control, showed no change in area therefore demonstrating the specificity of the phenotype. Additionally, overexpression of Dis3L2 throughout the wing imaginal disc (using 69B-GAL4) results in a significant reduction in proliferation, measured using phosphohistone H3 staining (Fig 3D), which in turn results in a reduction in the number of cells in the adult wing (Fig 3E). Taken together these data therefore confirm that Dis3L2 is a master regulator which plays a critical role in controlling developmental proliferation and ultimately tissue size without being limited by other accessory factors.

Fig 3

Overexpression of Dis3L2 results in a reduction in proliferation in the wing imaginal disc.

A) Western blotting of en-GAL4 driven Dis3L2 overexpression (dis3L2^OXP) wing imaginal discs confirms a 5-fold upregulation in Dis3L2 protein throughout the disc. Control consists of the parental controls with either the UAS or GAL4 insertion alone, UAS parent shown. n = 3, error bars represent SEM. B) Overexpression of Dis3L2 using en-GAL4 shows a significant reduction in the posterior area of the wing. Controls consist of parental controls to account for genetic background. n = 26–28, error bars represent 95% CI, **** = p<0.0001, ns = p>0.05. B’) Normalisation of posterior area of the wing to the anterior area shows a significant reduction in the posterior area of the wing. n = 26–28, error bars represent 95% CI, **** = p<0.0001. Overlay or representative control (blue) and Dis3L2 overexpression (orange, dis3L2^OXP) wings also shown. C) Overexpression of Dis3L2 in the posterior compartment of the wing imaginal disc (using en-GAL4) results in a reduction of the posterior area, represented as the posterior area normalised to the size of the anterior area of the tissue. The control group consists of; en-GAL4, UAS-GFPactin/+; to allow measurement and ensure that the en-GAL4 insertion and expression of GFP is taken into account. n = 26–33, error bars represent 95% CI, *** = p = 0.0008. C’) Representative images of control and Dis3L2 overexpression (dis3L2^OXP) wing discs when en-GAL4 is used to drive UAS-GFP expression and UAS-Dis3L2 in the case of Dis3L2 overexpression, or just UAS-GFP in control tissues. Scale bar = 50μm. A cartoon overlay of posterior compartment of control (blue) and overexpression (orange) wing imaginal discs is also shown. D) Overexpression of Dis3L2 throughout the wing imaginal disc (using 69B-GAL4) results in a reduction in the mitotic index of the wing imaginal disc. Control represents a combination of both parental controls (;;69B-GAL4 and;; UAS-Dis3L2). n = 6–10, error bars represent 95% CI, ** = p = 0.0076. E) Consistent with the mitotic index calculations, overexpression of Dis3L2 throughout the wing imaginal disc using 69B-GAL4 results in a reduction in the total number of cells in the wing. n = 13–22, error bars represent 95% CI, ***p = 0.0005.

RNA-sequencing reveals differential expression of pro-proliferative transcripts

As Dis3L2 regulates proliferation through its catalytic activity we hypothesised that it achieves this by degrading specific, growth promoting transcripts. To identify the RNAs that become misexpressed in dis3L2¹² wing discs, and that are responsible for driving the overgrowth phenotype, we performed RNA-sequencing on 120hr old dis3L2¹² and control wing imaginal discs in triplicate following rRNA depletion (see Methods for sample details). RNA-sequencing fastq files were then processed and aligned to the Drosophila genome (Flybase release 6.18; see methods for further details). Following differential expression analysis using individual replicate comparisons and stringent filtering criteria we identified 501 genes that showed differential expression in dis3L2¹² wing imaginal discs (Fig 4A). Of these, 344 were upregulated >1.34 fold and 157 were downregulated >1.34 fold (S3A Fig). The top 30 upregulated transcripts are shown in Fig 4B. Interestingly, transcripts differentially expressed in dis3L2¹² wing imaginal discs have a significantly shorter 3’ UTR (446bp) than the global average (598bp) whilst the average lengths of the 5’UTR and CDS do not differ (S4B Fig) which is congruent with findings in S. pombe [3].

Fig 4

RNA-seq analysis of transcripts misregulated in dis3L2 mutants compared to controls.

A) 501 transcripts (red dots) were determined as differentially expressed between dis3L2¹² and control wing imaginal discs following stringent filtering criteria. B) Top 30 upregulated transcripts and their replicate FPKMs in control (blue) and dis3L2¹² (red) wing discs. C) 5 out of 6 selected transcripts showed significant increases in expression by qRT-PCR in both the sequenced samples and fresh validation samples. n = 6, error bars represent SEM, **** = p<0.0001, ** = p<0.01, ns = p>0.05. D) idgf1, 2 and 3 are upregulated at the post-transcriptional as well as the transcriptional levels in the dis3L2 null mutant compared to controls. qRT-PCR was used to detect the pre-mRNA and mRNA transcripts. n = 6, error bars represent SEM, p<0.05 for all.

Consistent with previous findings we observed changes in expression in a variety of mRNAs and non-coding RNAs (ncRNAs) [2,11–14]. Regarding these ncRNAs that showed differential expression, we observed an increase in RNaseMRP:RNA including reads mapping to unprocessed RNaseMRP:RNA (S3B Fig) corroborating findings in Drosophila S2 cells and whole flies [13] suggesting that removal of unprocessed RNaseMRP:RNA is a global function of Dis3L2. This is also likely to be a conserved function as similar findings were observed in mouse embryonic fibroblasts [12]. Other ncRNAs found to be significantly upregulated include asRNA:CR45208, CR40461 and asRNA:CR42871; the functions of these are at present unknown.

Gene ontology (GO) analysis of the upregulated protein-coding transcripts revealed an enrichment of genes involved in carbon metabolism, symporters and glycosidases; whilst 33% of downregulated transcripts are membrane proteins (S3C Fig). Remarkably, the top 20 upregulated transcripts include 3 imaginal disc growth factors (idgf1/2/3), characterised in the ‘Glycosidase’ GO term, which show 74.9, 10.3 and 6.6-fold increases respectively. In Drosophila there are six members of the Idgf family (Idgf1-6); of these only idgf1, 2 and 3 are sensitive to the dis3L2 mutation with idgf4, 5 and 6 showing no changes in expression (S3D Fig) indicating specific regulation. In addition to the selected Idgfs, another mRNA encoding the conserved growth factor miple1 increased in expression by 22.3-fold. In total 6 transcripts were selected for validation by qRT-PCR due to their known roles in cell proliferation as well as syt4 which showed the largest increase in expression. qRT-PCR confirmed that all but syt4 showed significant upregulation in both the samples sent for sequencing and in three additional replicates of dis3L2¹² and dis3L2^wt wing imaginal discs (Fig 4C). To confirm the upregulation was specific and not caused by an off-target effect of the CRISPR process we assessed the levels of the 5 upregulated transcripts (excluding syt4) in dis3L2¹² hemizygote wing discs. Significant upregulation of each transcript was also observed in these tissues, confirming their Dis3L2 sensitivity (S4C Fig).

Idgf2 drives wing overgrowth in dis3L2¹² flies

In order to narrow down the mRNAs sensitive to dis3L2 mutations, we used additional, publicly available data sets derived from different Drosophila tissues. Re-analysis of RNA-sequencing data from dis3L2 null mutant testes [11] showed that 10% of transcripts differentially expressed in the mutant wing imaginal discs were also misexpressed in mutant testes (S4D Fig) showing that transcripts can be both globally sensitive to Dis3L2 or show tissue specificity. Upregulation of idgf1, 2 and 3 transcripts but not miple1 and cas was observed in both tissues suggesting intrinsic sensitivity to Dis3L2. If Dis3L2 is able to directly degrade idgf1, 2 and 3 we would expect these RNAs to be bound by Dis3L2. Since CLIP-seq analysis, which can detect the binding of proteins to RNAs, is challenging to perform on the small amounts of imaginal disc tissue dissected from Drosophila larvae, we turned to an existing publicly available data from Drosophila S2 cells [13]. These data show that idgf1, idgf2 and idgf3 transcripts are all pulled down with a catalytic dead Dis3L2 in Drosophila S2 cells [13], demonstrating that they are physically associated with Dis3L2 and that Dis3L2 can directly target and degrade these transcripts. We also assessed the transcriptional contribution towards the increased levels of idgf1, 2 and 3 by assessing changes in pre-mRNA expression. All three transcripts also showed pre-mRNA increases in the dis3L2 mutant (Fig 4D), therefore it is likely that the increase observed in dis3L2¹² tissues is a consequence of a combination of transcriptional and post-transcriptional effects.

Since the transcripts encoding idgf1, 2 and 3 appear to be globally sensitive to Dis3L2 and directly bound to the enzyme we used the power of Drosophila genetics to assess their individual contribution towards the overgrowth phenotype in dis3L2¹² animals. To achieve this, we used UAS-RNAi lines to idgf1, 2 or 3 each of which showed a knockdown of >90% at the RNA level when driven ubiquitously (by Tubulin-GAL4) with no obvious phenotypic defects (S5A Fig). As in our previous rescue experiments, we again made use of the en-GAL4 driver to deplete the levels of each idgf transcript individually in a dis3L2¹² mutant background. If any of these idgfs were individually responsible for the overgrowth phenotype we would expect individual knockdown to rescue the overgrowth phenotype resulting from the dis3L2 null mutation specifically in the posterior region as is observed when UAS-Dis3L2 is driven by en-GAL4 (Figs 5A and S2C). Using this approach to knockdown idgf1 or idgf3 in the dis3L2 mutant background had no effect on the posterior area of the tissue (Fig 5A). In contrast, depletion of idgf2 resulted in a complete and specific rescue of the posterior area of the wing (Fig 5A). Importantly, knockdown of idgf2 in a dis3L2 wild-type background has no effect on the area of the tissue (Fig 5B). Consistent with a role for Dis3L2 in regulating Idgf2, re-expression of Dis3L2 specifically in dis3L2¹² mutant wing discs also results in a reduction of idgf2 mRNA (S5B Fig). Further, levels of idgf2 mRNA were also significantly increased in wing imaginal discs dissected from a publicly available independent line carrying a CRISPR engineered catalytic dead dis3L2 mutation [13] (S5C Fig).

Fig 5

Idgf2 drives tissue overgrowth in dis3L2 mutant animals.

A) Knockdown of idgf2 (purple) but not idgf1 (pink) or idgf3 (pale purple) in the posterior region (en-GAL4) of dis3L2 mutant wing imaginal discs rescues the wing overgrowth phenotype to the same extent as expressing Dis3L2 itself (green). n = 16–34, error bars represent 95% CI, **** = p<0.0001, ns = p>0.05. Dotted lines represent the average area of the posterior region of mutant and control wings. B) Knockdown of idgf2, using en-GAL4, in a dis3L2^wt background has no effect on wing area. n = 16–18, error bars represent 95% CI, **** = p<0.0001, ns = p>0.05. C) Overexpression of Idgf2 throughout the wing imaginal disc using the 69B-GAL4 driver (purple) results in overgrowth of the wing to the same extent as observed when dis3L2 is knocked down using the same method (red). n = 18–35, error bars represent 95% CI, **** = p<0.0001. D/D’) Assessment of wing cell number (D) and wing cell size (D’) when Idgf2 is overexpressed throughout the wing using 69B-GAL4 demonstrates an increase in cell number. n = 19–22, error bars represent 95% CI, **** = p<0.0001. E) Idgf2 overexpression (with 69B-GAL4) results in an increase in wing imaginal disc area. n = 9–13, error bars represent 95% CI, ****p<0.0001. F) Consistent with an increase in cell number Idgf2 overexpression wing imaginal discs (UAS-Idgf2/+; 69B-GAL4/+) show increased proliferation to a level consistent with that observed in dis3L2 deficient wing imaginal discs (UAS-dis3L2^RNAi/+; 69B-GAL4/+). n = 9–13, error bars represent 95% CI, * = p<0.05, scale bar = 50μm.

To confirm that increased expression of Idgf2 alone is sufficient to drive tissue overgrowth and proliferation in-vivo we generated a UAS-Idgf2 transgenic line which resulted in a significant increase idgf2 expression when expressed throughout the wing imaginal disc (using 69B-GAL4) (S5D Fig). Overexpression of Idgf2 throughout the wing imaginal disc resulted in significant wing and wing imaginal disc overgrowth (as a result of increased proliferation in the wing imaginal disc and subsequent increased number of cells in the adult wing) to a level not different from that observed following dis3L2 depletion using the same 69B-GAL4 driver (Fig 5C–5F).

Loss of DIS3L2 in HEK-293T cells drives proliferation and upregulates the PI3-Kinase pathway

Given the lack of knowledge about the mechanism whereby Idgf2 drives proliferation in Drosophila we turned to human cell culture to identify the signalling pathway activated in DIS3L2 deficient cells. Previous work in HeLa cells demonstrated that reduction in DIS3L2 levels results in an increase in cell number [9]. Given the association between DIS3L2 and the kidney tumour, Wilms’ Tumour, we tested whether the loss of DIS3L2 was sufficient to promote proliferation in HEK-293T cells, which are a more physiologically relevant, embryonic kidney derived cell line. Using siRNA against DIS3L2 we achieved a maximal knockdown of 91.8% (72hrs post transfection) with a knockdown of >85% between 48 and 96 hours post transfection (144hrs post transfection a 62% knockdown was still present) (S6A Fig). During this period we observed an increase in cell number in the DIS3L2 knockdown cells compared to a scrambled siRNA control, demonstrating a conserved role for controlling proliferation in a clinically relevant cell type (Fig 6A). Interestingly, the hyperplasia resulting from DIS3L2 depletion has some tissue specificity as the same experiments in the osteosarcoma cell line U-2 OS showed no phenotype (Figs 6B and S6B).

Fig 6

DIS3L2 depletion in HEK-293T cells results in PI3-K induced hyperplasia.

A) Knockdown of DIS3L2 in HEK-293T cells confers a growth advantage compared to a scrambled control. n = 6, error bars represent 95% CI, * represent significant differences where error bars to not overlap. B) Knockdown of DIS3L2 in U-2 OS cells has no effect on cell number compared to a scrambled control. n = 6, error bars represent 95% CI, * represent significant differences where error bars to not overlap. C) Representative image and quantification of key growth promoting phospho-proteins in HEK-293T cells treated with siDIS3L2 (DIS3L2^KD) or siScrambled (Scrambled). n = 5, error bars represent SEM. D) Representative image and quantification of three different 4E-BP phosphorylation sites in HEK-293T cells treated with siDIS3L2 (DIS3L2^KD) or siScrambled (Scrambled). n = 3, error bars represent SEM. E) Treatment of DIS3L2^KD HEK-293T cells with Wortmannin (250nM) results in a rescue of the proliferation phenotype to that of a scrambled control with or without Wortmannin treatment (DMSO). Note that the growth of the scrambled control is not affected by this dose of Wortmannin. n = 4, error bars represent 95% CI, * represent significant differences where error bars to not overlap. F) Western blot showing the increased levels of pAKT (T308), pPRAS40 or p4E-BP (T37/46) in DIS3L2^KD HEK-293T cells 72hrs post transfection is lost upon wortmannin treatment. Hatched bars represent samples treated with wortmannin, n = 5, error bars represent SEM. ** = p<0.01, * = p<0.05.

Having confirmed the conservation of the phenotype between Drosophila and human cells, we set out to identify the molecular pathway controlled by DIS3L2 to regulate proliferation. We first made use of publicly available RNA-sequencing data from HEK-293T cells that conditionally express a catalytically dead DIS3L2 [14] and identified 111 differentially expressed transcripts. Gene ontology analysis of these transcripts indicated the potential enrichment of the PI3 Kinase (PI3-K) pathway (the only significant hit with an enrichment score of 1.93). This would be consistent with previous data showing that ectopic expression of an Idgf2 orthologue, CHI3L1, activates the AKT pathway in a variety of human cells [18–20]. To test the effect of depletion of DIS3L2 on the PI3-K/AKT pathway we used Western blotting to assess the phosphorylation status of AKT (T308 and S473) in addition to downstream pathway members. We also assessed the phosphorylation status of p42/44 MAPK, another crucial, conserved growth mediator also shown to be activated by ectopic CHI3L1/2 expression [21–23]. This demonstrated that loss of DIS3L2 did not affect MAPK phosphorylation but results in increased phosphorylation of pAKT at T308, and a complementary increase in phosphorylation of one of its targets, pPRAS40 (Fig 6C). T308 is known to be phosphorylated by PDK1 following PI3-K activation [24], whereas S473, which shows a partial but inconsistent increase, is phosphorylated by mTORC2 [25].

Having identified an apparent activation of PI3-K/AKT signalling in DIS3L2 deficient cells we asked if mTORC1 was activated, as might be expected for PI3-K dependent growth. We assessed the phosphorylation status of the mTORC1 target, 4E-BP on 4 conserved sites. This approach demonstrated that increased phosphorylation on S65 and T37/46 was observed in DIS3L2 knockdown cells (Fig 6D). However, we could not confirm increased T70 phosphorylation. To ask if the observed changes in phosphorylation status represented post-translational pathway activation or protein upregulation, we assessed changes in total levels of AKT, PRAS40 and 4E-BP. In all cases we saw changes in phosphorylation were over and above changes in steady state levels of the proteins (S6C Fig). However, due to some technical variation it is unclear as to whether the changes are solely driven by increased phosphorylation and it remains possible that these observations could be caused a combination of protein upregulation and post-translational modification. To phenotypically assess if the PI3-K pathway is responsible for driving overgrowth in DIS3L2 knockdown HEK-293T cells we treated them with the PI3-K inhibitor wortmannin. Whilst the addition of 250nM wortmannin to control cells did not appear to affect growth, wortmannin treatment of DIS3L2 knockdown HEK-293T cells resulted in a complete rescue of overgrowth, demonstrating a growth profile similar to that of the scrambled control with or without wortmannin (Fig 6E). Wortmannin activity was confirmed by assessing the levels of pAKT (T308), pPRAS40 and p4E-BP (T37/46) by Western blot which showed a specific reduction in phosphorylation in DIS3L2^KD HEK-293T cells following wortmannin treatment (Figs 6F and S6D). Taken together, although further work is required to elucidate the manner of PI3-Kinase pathway upregulation, this data demonstrates the first example of a mechanism through which DIS3L2 regulates proliferation in human cells, and may be consistent with the overgrowth induced by the orthologues of Idgf2.

Increased proliferation in dis3L2¹² wings is wortmannin-sensitive

Having identified a role for the PI3-K/AKT pathway in DIS3L2 knockdown HEK-293T cells we asked if the same pathway was mediating growth in the Drosophila wing imaginal discs. To address this, we fed wortmannin to larvae at concentrations of 5μM and 10μM at various stages during their development. At these optimised concentrations, the wild-type larvae are not affected. Whilst the presence of wortmannin from 24, 48 or 72hrs into development had no effect on dis3L2^wt wing area, dis3L2¹² wings were significantly smaller than those fed the vehicle control food (containing DMSO) (Fig 7A–7C). Although the rescue of wing area was not complete, we saw a significant reduction in the overgrowth suggesting that Dis3L2-mediated control of growth occurs in a conserved manner at least partially through a PI3-Kinase sensitive network. Consistent with a role for Idgf2 in driving the overgrowth phenotype in dis3L2¹² animals, the feeding of wortmannin to flies overexpressing Idgf2 (throughout the wing imaginal disc using 69B-GAL4) also results in a comparable reduction in tissue area (Fig 7D). Additionally, and congruent with our HEK-293T cell data, we observe increased phosphorylation of AKT at T342 (homologous to T308) in both dis3L2¹² larvae and those with ubiquitous Idgf2 overexpression demonstrating the conservation of Dis3L2-mediated growth control (Fig 7E). However, it is important to note that we were unable to assess total AKT levels in our mutant/overexpression animals and therefore further work is required to identify the mechanism behind the increased phosphorylation of AKT. Finally, although wing area is reduced in dis3L2¹² flies following wortmannin treatment, the addition of wortmannin has no effect on the overexpression of idgf2 within the wing imaginal disc showing that Idgf2 overexpression lies genetically upstream of the activation of the PI3-K pathway in these tissues (Fig 7F). Together these data are consistent with a role for Idgf2 in driving the wortmannin sensitive pathway to promote proliferation and tissue overgrowth in dis3L2¹² tissues. Further work is required to investigate if Idgf2 directly activates the PI3-Kinase network in Drosophila together with the nature of the pathway activation; the identification of the Idgf2 receptor is crucial to this concept. Nevertheless, we have, for the first time, uncovered a conserved mechanistic pathway whereby Dis3L2 regulates cell proliferation in both Drosophila and human cells.

Fig 7

PI3-K is involved in driving proliferation in dis3L2¹² mutant wings.

A-C) Feeding of Wortmannin to Drosophila larvae at 5μM or 10μM results in a significant reduction specifically in dis3L2¹² wings and not dis3L2^wt wings. Developing larvae were cultured on Wortmannin or control (DMSO) containing food from (A) 24, (B) 48 or (C) 72 hours after egg lay (AEL). n = 16–58, error bars represent 95% CI, *** = p<0.001, **** = p<0.0001. D) Feeding of Wortmannin to larvae at 10μM results in a significant reduction of Idgf2 overexpression (69B>idgf2) wing area but not control (69B-GAL4 parental) wings. Developing larvae were cultured on Wortmannin or control (DMSO) containing food from 48 hours AEL. n = 20–33, error bars represent 95% CI, **** = p<0.0001. E) Assessment of AKT phosphorylation at T342 in dis3L2^wt, dis3L2¹², and Idgf2 overexpression (driven by Tub-GAL4) 120hr old L3 larvae shows increased T342 phosphorylation in L3 larvae mutant for dis3L2 and those with ubiquitous Idgf2 overexpression. n = 6–10, error bars represent SEM, * = p = 0.0121, *** = p = 0.0003. F) Addition of 10μM Wortmannin does not affect idgf2 overexpression in dis3L2¹² wing imaginal discs showing Idgf2 is upstream of the Wortmannin sensitive pathway. n = 3–4, error bars represent SEM, ** = p<0.01.

Dis3L2 induced overgrowth in Drosophila requires upregulation of the growth factor Idgf2

In this study we have used a new dis3L2 null mutant to identify Idgf2 as the factor responsible for driving proliferation and overgrowth in dis3L2 loss of function Drosophila tissues. Using RNA-sequencing data generated in this study, together with our analysis of published data, it is clear that idgf2 expression is sensitive to Dis3L2 in a variety of Drosophila tissues. Crucially, idgf2 mRNA has been shown to co-precipitate with a catalytic dead Dis3L2 in Drosophila embryonic S2 cells, suggesting Dis3L2 is able to directly degrade idgf2 mRNA. Interestingly, idgf2 mRNA also increases in levels, although to a lesser extent, in testes mutant for the terminal uridylyl transferase tailor [11], indicating that idgf2 may be targeted to Dis3L2 by 3’ uridylation. Further, re-expression of Dis3L2 specifically in the wing disc results in specific reduction in idgf2. Therefore, although further work is required to assess the ways in which Dis3L2 mediates clearance of idgf2 transcripts, these analyses provide strong evidence that Dis3L2 functions to control proliferation in Drosophila by regulating idgf2 expression. Interestingly, we also observe an increase in idgf2 pre-mRNA suggesting an additional increase at the transcriptional level. This therefore suggests that the increase in idgf2 in dis3L2 mutant tissues is a combination of direct and indirect effects which subsequently results in the overgrowth of the tissue.

Although Drosophila Idgf2 is the best molecularly characterised of the Idgfs in that its crystal structure has been determined [26], the downstream pathway it activates remains elusive, although data presented here suggests that Idgf2 could promote PI3-K signalling to drive proliferation. For example, the fact that both Idgf2 overexpression and Dis3L2 mutant larvae show increased phosphorylation of AKT at T342, demonstrate wortmannin-sensitive overgrowth, and that idgf2 expression remains elevated in dis3L2 mutant tissues treated with wortmannin suggests that Idgf2 lies upstream of the wortmannin-sensitive pathway. We have shown that increased Idgf2 is sufficient to drive proliferation and tissue growth and results in increased AKT phosphorylation at T342 which provides in-vivo evidence to support previous findings that Idgf2 promotes proliferation of Drosophila wing disc cells in culture [27,28]. Whilst the wortmannin data suggests the wing overgrowth is indeed PI3-K sensitive it is important to note that due to technical difficulties regarding the small size of the wing imaginal discs these preliminary conclusions are drawn on system-wide changes across the larvae. Further work would therefore be required to identify of the nature of increased pathway activity in addition to specifically addressing the signalling mechanisms within the wing imaginal disc. Interestingly, Blanco et al showed that Idgf2 increases in expression by 4-fold during early stages of tissue regeneration [29]. Further, it has recently been shown that Idgf2 protects imaginal disc cells (C1.8) in culture from apoptosis and that this cytoprotection is associated with the induction of genes associated with energy metabolism and innate immunity [27]. Our SUnSET labelling, which shows increased translation in dis3L2 mutant cells, would support the idea that these cells are more physiologically active than normal cells. It is therefore possible, that under normal conditions, Dis3L2 functions to restrain Idgf2 signalling, however, in its absence the wing disc cells are ‘primed’ in a more proliferative state to facilitate the repair of damaged tissue or protect the cells from death.

Idgf2 is a member of the conserved Chitinase-like protein (CLP) family and orthologous to CHI3L1 and CHI3L2. Consistent with our findings, both CHI3L1 and CHI3L2 have been shown to drive proliferation in a variety of human cell types [18,20–23,30] in addition to being associated with numerous chronic inflammatory diseases and cancers including glioblastoma, non-small cell lung cancer and colon cancer. Interestingly, in the case of colorectal cancer, downregulation of DIS3L2 (as a consequence of knockdown of lncRNA AC105461.1) enhances the proliferation and stem-cell like properties of cells of the colorectal cancer line SW480 [31]. In line with the data presented here, ectopic CHI3L1 expression has been shown to stimulate AKT phosphorylation in a variety of cell types [18–20,30,32]. However, CHI3L1 and CHI3L2 are expressed at very low levels in immortalised cell lines and we were unable to reliably detect an increase in CHI3L1 or CHI3L2 transcripts in our HEK-293T cells. Therefore, it is also possible that another factor may be responsible for the hyperplasia observed in our DIS3L2-deficient HEK-293T cells.

DIS3L2 represses PI3-K/AKT pathway activity in HEK-293T cells

DIS3L2 loss of function mutations have been implicated Perlman Syndrome and Wilms’ tumour of the kidney [9]. To assess the role of DIS3L2 in human kidney cells we depleted DIS3L2 in HEK-293T cells and, as in Drosophila, we observed strong increases in proliferation and identified a potential role for the PI3-Kinase in driving this phenotype. The role of DIS3L2 in human cells was also shown to have some specificity since the overgrowth phenotype is not observed in following DIS3L2 knockdown in the osteosarcoma cell line U-2 OS. Although the PI3-K pathway is active in both these cell lines, there are many possible reasons why the two cell lines do not behave in the same way. For example, the differences could also be due to the tissue of origin of the cell line, or could be due to HEK-293T being virally immortalised rather than cancerous and therefore this genetic difference may also be the cause of the different behavior of the two cell lines. Mammalian target of rapamycin (mTOR), is a downstream mediator in the PI3-K signalling pathway and is an essential serine/threonine kinase which is aberrantly activated in human osteosarcoma [33,34]. As this activation of mTORC is often independent of upstream signalling, including PI3-K, this could provide an explanation for the lack of influence of DIS3L2 on proliferation in osteosarcoma cells. Alternatively, given that Perlman Syndrome and Wilms’ tumour are early, developmental diseases is could be that DIS3L2 is essential to maintain proliferation early in development. U-2 OS cells are from an adolescent whilst HEK-293T cells are embryonic and therefore this could influence the effect of loss of DIS3L2. This would be congruent with our previous work showing that Dis3L2 is critical to control proliferation during early development in Drosophila [1].

We observed increases in AKT/mTORC1 activity following the loss of DIS3L2 in HEK-293T cells, providing the first mechanistic explanation for DIS3L2 induced overgrowth in mammalian cells, although further work is required to better understand that nature of pathway activation. Interestingly, we saw increased phosphorylation of the PDK1 sensitive T308 site of AKT, however, we did not see consistent increases in S473 phosphorylation, catalysed by mTORC2 [25]. T308 has been shown to be sufficient for AKT activity, although its activity is reduced without S473 phosphorylation [35,36]. Consistent with our data, in both Drosophila and mouse models it has been shown that phosphorylation of the hydrophobic motif (S473 in humans and mice, S505 in Drosophila) is important for FOXO regulation but is not required for mTOR activation and regulation of T342 phosphorylation in Drosophila is important for growth control [37–40]. Whilst the lack of S473 activation may due to technical issues, it may also explain the controlled tissue growth we see in Drosophila. For example, the partial activation of AKT may be sufficient to drive increased growth through TORC1, however, without S473 activation, and subsequent FOXO inhibition, some control over proliferation is retained. Whilst we observe increased phosphorylation of AKT at T342 in-vivo in both Dis3L2 null and Idgf2 overexpressing animals, the lack of S473 phosphorylation in our experiments in HEK-293T cells is also consistent with the lack of activation of the corresponding site in Drosophila (S505) following ectopic Idgf2 expression in C1.8 cells [27].

Our conclusion that the PI3-K pathway contributes towards loss of DIS3L2 induced overgrowth is strengthened by the observation that the increased proliferation in DIS3L2 deficient cells was specifically inhibited following wortmannin treatment. These findings are also congruent with our re-analysis of RNA-seq data from DIS3L2 catalytic dead HEK-293T cells [14] which, in our hands, shows the only significantly differentially expressed pathway to be the PI3-K pathway. This apparent role for PI3-K in mediating Dis3L2-regulated growth appears to be conserved as we also present preliminary data in our Drosophila model demonstrating wortmannin-sensitivity and increased AKT phosphorylation. The mechanism behind the increased phosphorylation and whether Idgf2 directly activates this pathway, or there is co-operation with other pathways in Drosophila requires further work and remains under investigation.

How is Dis3L2 recruited to its RNA targets?

At present the molecular mechanisms whereby Dis3L2 specifically regulates the expression of idgf2 are unknown. One hypothesis is that sequences or structures within the mRNA itself render it particularly susceptible to degradation by Dis3L2. Previous work in human and Drosophila cells has identified a role for Dis3L2 in regulating RNA polymerase III transcripts [12–14] which have U-rich 3’ ends as a result of pol III terminators. Interestingly, MEME [41] analysis of the 3’ UTRs of mRNAs upregulated in dis3L2¹² mutant wing discs which also co-precipitate with Dis3L2 [13] show a significant enrichment of a U-rich motif (40% submitted transcripts), and a CA-rich motif (14.1% of submitted transcripts) which are not enriched in a control data set (mRNAs that do not change in expression or co-precipitate with Dis3L2 in S2 cells) (S4E Fig). Given the preference of Dis3L2 in degrading U-rich regions this could demonstrate an intrinsic mechanism facilitating Dis3L2-mediated decay of a specific set of mRNA substrates. Further, the control data set showed enrichment of a G-rich motif which was not identified in the list of likely Dis3L2 substrates (S4F Fig). The inability of Dis3L2 to degrade G terminated RNAs [13], together with the fact that G-rich regions form stabilising G-quadruplex structures further strengthens the validity of our target dataset. Finally, transcripts with shorter 3'UTRs appear to be more sensitive to Dis3L2 activity (446nt vs 598nt; S4B Fig) which is consistent with data from S. pombe [3], however, the reason behind this remains unknown. Consistent with these findings, idgf2 has a very short 3’ UTR at 50bp in length and although it does not contain an extended U-rich motif as identified above, it does contain an AU-rich element which has been shown to target transcripts for 3’-5’ decay by both the exosome and Dis3L2.

Whilst the data presented here demonstrate a novel, and potentially conserved, mechanism through which Dis3L2 mediated decay of idgf2 is required to control tissue growth, the downstream signalling pathway activated by Idgf2 remains elusive. However, it appears that the involvement of the PI3-K pathway in inducing Dis3L2-mediated overgrowth is conserved between Drosophila and human HEK-293T cells given our identification of increased AKT phosphorylation in both models. Further understanding of these pathways is critical to understanding not only the DIS3L2 regulated conditions but the many other conditions also driven by the increased expression of the CLP family of proteins. Exploring the endogenous role of CLP proteins in tissue culture cells is difficult due to their low levels of expression and therefore our in-vivo Drosophila model is an exciting and appropriate system to further unlock this largely unknown pathway of tissue growth and proliferation.

Drosophila husbandry

Fly stocks were cultivated on standard media in uncrowded conditions at 25°C unless otherwise stated. The following stocks were obtained from Bloomington Stock Center: Tub-GAL4 (P{w^+mC = tubP-GAL4}LL7 originally from stock 5138, y¹ w^*;; P{w^+mC = tubP-GAL4}LL7/TM6b,GFP), act5C-GAL4 (stock 4414, y¹ w^*; P{w^+mC = Act5C-GAL4}25FO1/CyO, y⁺;), nub-GAL4 (stock 25754; P{UAS-Dcr-2.D}1, w¹¹¹⁸; P{GawB}nub-AC-62), 69B-GAL4 (stock 1774; w*;; P{GawB}69B), UAS-idgf1^RNAi (stock 57508, y¹sc^*v¹; P{TRiP.HMC04823}attP40), UAS-idgf2^RNAi (stock 55935, y¹sc^*v¹; P{TRiP.HMC04223}attP40), UAS-idgf3^RNAi (stock 67226, y¹sc^*v¹; P{TRiP.HMC06327}attP40), Df(3L)Exel6084 (stock 7563, w¹¹¹⁸;; Df(3L)Exel6084, P{XP-U}Exel6084/TM6B). Stocks obtained from the Vienna Drosophila Resource Center were: UAS-dis3L2^RNAi (stock v51854, w¹¹¹⁸; P{GD9240}v51854; and stock v100322,;; P{KK105902}VIE-260B) and Dis3L2^CD (stock 312503). en-GAL4 (A kind gift from Paul Martin,; engrailed-GAL4,UAS-GFPactin/Cyo;). To overexpress Dis3L2 the stock w*;; P{GSV3}GS6090/TM6 was purchased from the Kyoto Stock Center (stock 200902), however, a mutation outside the Df6084 locus caused homozygous lethality; this was repaired by recombination.

Generation of CRISPR mutants

A gRNA to the first exon of dis3L2 was cloned into the Bbs1 site of pCFD3-dU6:3. The resulting vector was then sent to BestGene Inc for injection into vas-Cas9 embryos (BDSC 51323 y¹ M{vas-Cas9}ZH2A w¹¹¹⁸). Potential mutant chromosomes were balanced and subject to PCR screening using the gRNA as a forward primer (see S1 Table for all primers/gRNA used in this study). Potential mutant stocks were sequenced at Eurofins Genomics. An 8bp frame shift mutation was identified and its lack of Dis3L2 confirmed by Western blotting. This line was named dis3L2¹².

Western blotting and generation of anti-dDis3L2 antibody

A polyclonal antibody was raised against the N-terminal tagged Dis3L2 peptide consisting of the first 198 amino acids of the Drosophila protein. pDNR plasmid-B527503, containing the cDNA for Drosophila CG16940 RC gene (Isoform PC), was used as template for the amplification of the Drosophila dis3L2 gene in the construction of two N-terminal fusion proteins with different tags (GST-Dis3L2 and MBP-Dis3L2), that were expressed and purified in E. coli. The GST- and MBP-tagged proteins were used for the immunization in the goat and for the affinity purification of the anti-serum, respectively (by SICGEN, Portugal).

Western blots were performed in samples of adult females (x3), 120hr L3 larvae (x5) or wing imaginal discs (x60). Tubulin was used as a loading control on all blots. All blots apart from those assessing AKT T342 phosphorylation were blocked in Odyssey Blocking Buffer (PBS) (LI-COR cat. no. 927–40000) with antibody incubations performed in Odyssey Blocking Buffer containing 0.1% Tween. Mouse anti-Tubulin primary antibody (Sigma, cat. no. T9026) was used at 1:2000 dilution. Goat anti-Dis3L2 (produced in this study) was used at 1:2500. Anti-mouse, anti-rabbit and anti-goat fluorescent secondary antibodies were used at 1:20,000 (LI-COR Donkey anti-mouse IRDye 800CW (cat. No. 925–32212), Goat anti-rabbit IRDye 680RD (cat. no. 926–68071) and Donkey anti-goat IRDye 680RD (cat. no. 925–68074)). Anti-human DIS3L2 (hDIS3L2) was used at 1:500 (Novus Biologicals cat. no. NBP-1-84740). Anti-GAPDH (Abcam cat. no. ab8245) was also used at 1:10,000 as a human specific loading control along with the anti-mouse IRDye 800CW secondary antibody above. Blots assessing AKT T342 phosphorylation were washed in 0.1% TBS-Tween and blocking and antibody incubation steps performed in 5% BSA in 0.1% TBS-Tween. Anti-pAKT (T342) (Phosphosolutions cat. no. p104-342) was used at 1:500. Detection and quantification were performed using the LI-COR Odyssey FC imager and Image Studio (version 5.2) respectively. Uncropped blots are shown in S2 File.

Phospho-specific western blotting in HEK-293T cells

Blots were performed on cell pellets from the specified time post transfection or treatment. Samples were ran on 4–12% Novex gradient gels (Invitrogen cat. no NP0321BOX) or Mini-Protean TGX Stain-Free 4–15% gels (Bio-Rad cat. no. 4568084). All membranes were blocked in 3% BSA in TBS with antibody incubation in 3% BSA in 0.1% TBS-Tween. Primary antibodies used were as follows: Anti-pAKT (T308) (Cell Signalling cat. no. 1303S), anti-pAKT (S473) (Cell Signalling cat. no. 4060S), anti-AKT (Cell Signalling cat. no. 4685S) anti-pp42/44 MAPK (T202/Y204) (Cell Signalling cat. no. 9101S), anti-pPRAS40 (T246) (Cell Signalling cat. no. 2997S), anti-PRAS40 (Cell Signalling cat. no. 9644S), pS6-Ribosomal Protein (Cell Signalling cat. no. 2215S), anti-p4E-BP (T37/46) (Cell Signalling cat. no 2855S), anti-p4E-BP (S65) (Cell Signalling cat. no. 9451S), anti-p4E-BP (T70) (Cell Signalling cat. no. 5078S) and anti-4E-BP (Cell Signalling cat. no. 2610S) were all used at 1:1000. Mouse anti-Tubulin primary antibody (Sigma, cat. no. T9026) was used at 1:2000 dilution. Goat anti-Rabbit IRDye 680RD (LI-COR cat. No. 925–68071) and Donkey anti-mouse IRDye 800CW (cat. No. 925–32212) were used at 1:20,000. Detection and quantification were performed using the LI-COR Odyssey FC imager and Image Studio (version 5.2) respectively. Differential phosphorylation status was assessed by normalising signal in DIS3L2^KD cells to the respective scrambled control ran on the same gel. Uncropped blots are shown in S2 File.

Assessing lifespan

1 day old dis3L2^wt and dis3L2¹² flies were transferred into new food. Every 3–4 days flies were transferred into new food and the number of deceased flies were counted. This continued until all flies had deceased.

Measurement of wing and wing imaginal disc area

Adult flies were aged to between 1 and 2 days old for all wing measurement experiments. A single wing was cut from each fly and stored in isopropanol for 1 hour before being mounted in DPX (Sigma cat. no. 06522). All results shown are for male wings, however, female wings also showed the same phenotypes. Mounted wings were measured using Axiovision 4.7 on an Axioplan microscope (Carl Zeiss). Imaginal discs were dissected in PBS from 120hr old L3 larvae, aged using a 1 hour egg lay. Dissected discs were mounted in 85% Glycerol on Poly-L-Lysine slides. Imaginal disc area was measured using Axiovision 4.7 on an Axioplan microscope (Carl Zeiss). Salivary glands were prepared and imaged using the same methodology as used for imaginal discs.

Measurement of whole flies and larvae

Control and mutant female flies were left for 1 hour to lay on grape agar plates at 25°C. 24 hours later 20 L1 larvae were transferred into food vials and left to develop in uncrowded conditions. 5 vials (100 larvae total) were set up for each genotype. Eclosing adults were then aged to 1 day old, individually weighed and photographed. Specific, defined regions of each male fly were then measured using ImageJ (S7 Fig). Larval mass was calculated by washing 120hr L3 larvae (cultured as above) in PBS then weighing individually in an Eppendorf. Larval footprint was calculated from the weighed flies following 30 min incubation in 100% Methanol where the larvae elongate. Larvae were then photographed and their “footprint” was measured in pixels using ImageJ.

SUnSET labelling

Surface sensing of translation (SUnSET) was performed on dis3L2^wt and dis3L2¹² wing imaginal discs. Wing discs were dissected in batches of 30 and incubated in fully supplemented Shields and Sang M3 insect medium (Sigma-Aldrich, cat. no S8398) containing 2μg/ml Puromycin (Sigma-Aldrich, cat. no. P8833) for 1 hour at 25°C with rotation. Western blotting was performed to determine the levels of Puromycin incorporation with Tubulin as a loading control. Mouse anti-Tubulin primary antibody (Sigma-Aldrich, cat. no. T9026) was used at 1:2000 dilution. Mouse anti-Puromycin (clone 12D10) primary antibody (Merck Millipore, cat. no. MABE343) was used at 1:1000 dilution. Anti-mouse IRDye 800CW secondary antibody (LI-COR Biosciences, cat. no 926–32210) was used at 1:20,000 dilution to detect both primary antibodies. Each sample was run in parallel on two gels/membranes so the Tubulin band could be distinguished from Puromycin containing peptides. Quantification of Tubulin (46kDa) and Puromycin peptides (from smallest size visible to 240kDa) was achieved using LI-COR Biosciences Image Studio software. In each case Puromycin signal in dis3L2¹² tissues was compared to dis3L2^wt samples ran on the same gel. Uncropped blot is shown in S2 File.

Fertility assays

Individual virgin, 3 day old male flies of the test genotype were crossed to 2 virgin dis3L2^wt females. Flies were left for 48 hours to mate, and then removed. Eclosing progeny were counted for 7 days. In all crosses using dis3L2¹² homozygous and hemizygous male parents, many eggs were laid by the dis3L2^wt females but none were fertilised.

Generation of UAS-dis3L2 and UAS-idgf2 lines

Drosophila dis3L2 was amplified from pDNR-DUAL (DGRC clone BS27503) using two primer pairs; (1) PC which amplified the full coding region of dis3L2 capable of encoding both isoforms and (2) PA which only amplified the coding region of the shorter isoform (PA). These were cloned into the AgeI and XbaI sites of pUASP-attB. Constructs were then sent to BestGene Inc for injection into a recipient line with an attP site at 51C1 (BDSC 24482; M{3xP3-RFP.attP’}ZHC51C). Lines where the insert was successfully integrated were confirmed by PCR and balanced. To produce the UAS-dis3L2^ND line site-directed mutagenesis was performed using the Agilent Quickchange Lightning mutagenesis kit (Agilent, cat. No. 210518), and primers containing the G1738A mutation (D580N) on the pUASP-attB vector containing the PC insert. Human DIS3L2 (Ensembl isoform 202) was amplified from oligo(dT) primed cDNA produced from hFOB cells (ATCC CRL-11372) and cloned into the NotI and SpeI sites of pUASP-attB. Injection and subsequent processing were performed as above. UAS-idgf2 was produced by amplifying idgf2 (CDS and 3’UTR) from cDNA from dis3L2^wt wing imaginal discs and subsequent cloning to the NotI and SpeI sites of pUASP-attB. The created vector was then injected into attP40 (y¹ w^67c23; P{CaryP}attp40) and balanced. The sequences of all primer sets used are described in S1 Table.

Mitotic index

Immunocytochemistry was performed essentially as described in [1] on 120hr L3 wing imaginal discs aged using 1 hour egg lay periods. Images were taken with a Leica SP8 confocal microscope. Anti-Phosphohistone H3 (Cell Signalling, cat no. 9701) was used at 1:300 dilution. Cy3-conjugated monoclonal Donkey anti-mouse IgG secondary antibody was used at 1:400 (Jackson ImmunoResearch, cat. no.715-165-151). The number of nuclei undergoing mitosis were counted using the ImageJ plugin DeadEasy MitoGlia [42]. The mitotic index was then calculated for each disc by dividing the number of cells in M phase by the area of the disc.

Wortmannin assay in Drosophila

Fresh food containing Formula 4–24 Instant Drosophila Medium (Carolina cat no. 173200), yeast and bromophenol blue was made each day with either the desired concentration of Wortmannin (5μM or 10μM) or an equal volume of DMSO. 4 hour egg lays were performed on grape agar plates and an equal number of larvae were transferred to Wortmannin or control food for each genotype at specific developmental time points (24hr, 48hr and 72hr after egg lay) then left to develop at 25°C. Consumption of the drug was confirmed by visualising the blue food in the gut. Wings were then cut and measured from eclosing adults as outlined above.

RNA-seq sample preparation and analysis

RNA was extracted from 60 wing imaginal discs dissected from 120hr L3 larvae. Three replicates of control and mutant were dissected. In these experiments, control samples were collected from a stock containing a 6bp mutation in dis3L2 that does not affect wing size or induce any other obvious phenotype (S4A Fig). Downstream qRT-PCR validation samples were collected from the dis3L2^wt stock which contains the wild-type sequence and were grouped together with the sequenced samples for downstream analysis (dis3L2^wt). To ensure accurate aging, 3 hour egg lays were used and wing discs were dissected in Ringers solution 120hrs after egg laying. RNA was extracted using a miRNeasy RNA extraction kit (Qiagen, cat. no. 217084), with on-column DNase digestion (Qiagen, cat. no. 79254). RNA concentrations were measured on a NanoDrop One spectrophotometer (Thermo Scientific). RNA integrity was assessed on an Agilent 2100 Bioanalyser.

400ng total RNA was depleted for ribosomal RNA as performed in [43] with the resulting RNA sent to Leeds Genomics for library preparation using the Illumina TruSeq Stranded protocol. Subsequent libraries were run in a paired-end sequencing run on a HiSeq 3000 generating between 36 and 44 million reads per sample. Raw RNA-sequencing files have been deposited in ArrayExpress. Accession number: E-MTAB-7451

Sequencing quality was assessed using FastQC c0.11.7 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and adapters were removed using Scythe v0.993b (https://github.com/vsbuffalo/scythe). Further quality control and read trimming was achieved using Sickle v1.29 (https://github.com/najoshi/sickle). The remaining high quality reads were mapped to the Drosophila melanogaster genome from Flybase (r6.18 [44]) using HiSat2 v2.1.0 [45]. Differential expression was completed and normalised FKPM values were generated using the Cufflinks pipeline [46]. Individual replicates from each condition showed good consistency (S1 File). Due to issues with statistical outputs from these pipelines we used individual replicate comparisons to minimise false positives when specifying that a gene is differentially expressed. All mutant replicates required a fold change of >1.34 compared to all control replicates. A fold change cut off of >1.34 was selected as this was the smallest fold change deemed significant from the Cuffdiff output. Alignment results and non-default parameters used during the analysis are shown in S1 File. Raw sequencing files from [11] were processed in the same analysis pipeline to allow direct comparison.

RNA extraction and qRT-PCR

RNA extractions were performed using a miRNeasy RNA extraction kit (Qiagen, cat. no. 217084), with on-column DNase digestion (Qiagen, cat. no. 79254). RNA concentrations were measured on a NanoDrop One spectrophotometer (Thermo Scientific).

For qRT-PCR, 1μg of total RNA was converted to cDNA in duplicate using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, cat. no. 4368814) with random primers. A control "no RT" reaction was performed in parallel to confirm that all genomic DNA had been degraded. qRT-PCR was performed on each cDNA replicate in duplicate (i.e. 4 technical replicates in all), using TaqMan Universal PCR Master Mix, No AmpErase UNG (Life Technologies, cat. no. 4324018) and an appropriate TaqMan assay (Life Technologies). For custom pre-mRNA assays, the pre-mRNA sequence of the desired target area was submitted to Life Technologies’ web-based Custom TaqMan Assay Design Tool as in [47] (S1 Table). rpL32 (rp49) was used for normalisation.

Human cell culture and growth curve analysis

HEK-293T and U-2 OS cells were cultured in Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12 –Gibco cat. no. 21331–020) supplemented with 10% foetal bovine serum (PAN Biotech, cat. no. P40-37500), 2mM L-Glutamine (Gibco cat. no. 25030–024) and antibiotics (100IU/mL penicillin, 100μg/mL streptomycin, Sigma Aldrich cat. no. 15140–122), at 37°C in a 5% CO₂ humidified incubator. For growth analysis of DIS3L2 knockdown, 3x10⁵ cells were plated in 6 well plates and transfected with 30pmol siRNA (DIS3L2 (Invitrogen cat. no. AM16708) or scrambled control (Invitrogen cat. no. AM4611)) using Lipofectamine RNAiMAX reagent (ThermoFisher cat. no. 13778030). Cell number was counted in triplicate every 24hrs for 144hrs. For Wortmannin treatment analysis cells 3x10⁵ HEK-293T cells were plated and treated with siRNA as above. 250nM Wortmannin or an equal volume of DMSO was then added 20, 44, and 68hrs, post-transfection. Cells were counted in triplicate every 24hrs following transfection.

Statistical tests

All statistical analyses were performed in either R v3.5.1 or GraphPad Prism 7. Two-sided two-sample t-tests were used to compare the means of single test groups to single control groups. If multiple comparisons were required, a one-way ANOVA was performed with a post-test to compare the means of each possible pair of samples.