Flies lacking Akt phosphorylation sites on both Tsc1 and Tsc2 are slightly lean.

<p>Triglyceride levels normalized to total body protein for Tsc1²⁹ homozygotes rescued by ubiquitous expression of Tsc1^WT (“WT”), Tsc1^S533A (“S533A”) or Tsc1^S533D(“S533D”), or flies homozygous for both the Tsc1²⁹ and Tsc2¹⁹² mutations rescued to viability by expression of both Tsc1^S533A and Tsc2^{T437A/S924A/T1054A/T1518A} (“double”). * indicates statistical significance (ttest = 0.01).</p

Flies lacking Akt phosphorylation sites on Tsc1 and Tsc2 are viable and normal in size.

<p>(A) Expression levels of myc-Tsc1 in fly lines homozygous for the Tsc1²⁹ mutation, rescued by ubiquitous expression of Tsc1^WT, Tsc1^S533A or Tsc1^S533D, or flies homozygous for both the Tsc1²⁹ and Tsc2¹⁹² mutations rescued to viability by ubiquitous expression of both Tsc1^S533A and Tsc2^{T437A/S924A/T1054A/T1518A} (“Tsc1^S533A,Tsc2^4A”). (B,C,D) Survival rates (B), pupation curves (C) and relative adult wing sizes (D) of animals seeded as L1 larvae under controlled growth conditions for genotypes w¹¹¹⁸ (“w¹¹¹⁸”), Tsc1²⁹ homozygotes rescued by ubiquitous expression of Tsc1^WT (“WT”), Tsc1^S533A (“S533A”) or Tsc1^S533D(“S533D”), or flies homozygous for both the Tsc1²⁹ and Tsc2¹⁹² mutations rescued to viability by ubiquitous expression of both Tsc1^S533A and Tsc2^{T437A/S924A/T1054A/T1518A} (“double”).</p

Knockdown of dSETD3 in the wing disc reduces growth.

<p><b>(A-B)</b> Knockdown of dSETD3 in the posterior compartment of the wing decreases tissue size. (A) Size of the posterior compartment normalized to size of the control anterior compartment. (B) Cell size in the posterior compartment was determined by counting the number of cells via their trichomes in a defined area, and then calculating the inverse–ie area per cell. Animals were raised at 25°C, 10 wings per genotype were used for quantifications. Error bars: standard deviation (SD); *** <i>t</i> test < 0.001 <b>(C)</b> Knockdown of dSETD3 in Drosophila S2 cells does not change mTORC1 activity. dSETD3 was knocked-down by treating S2 cells with 2 independent dsRNAs for 5 days, and mTORC1 activity was assayed via immunoblotting for phosphorylation of the direct target S6K. The two lanes per sample represent biological duplicates. <b>(D)</b> S2 cells treated with 3 different dsRNAs against dSETD3 do not show a change in cell proliferation rates. S2 cells were treated with dsRNAs for 5 days and plated freshly at the same cell density. Cell number was then counted every 24 hours for 5 days.</p

Phenotypic characterization of SETD3 knockout Drosophila

<div><p>Lysine methylation is a reversible post-translational modification that affects protein function. Lysine methylation is involved in regulating the function of both histone and non-histone proteins, thereby influencing both cellular transcription and the activation of signaling pathways. To date, only a few lysine methyltransferases have been studied in depth. Here, we study the <i>Drosophila</i> homolog of the human lysine methyltransferase SETD3, CG32732/dSETD3. Since mammalian SETD3 is involved in cell proliferation, we tested the effect of dSETD3 on proliferation and growth of <i>Drosophila</i> S2 cells and whole flies. Knockdown of dSETD3 did not alter mTORC1 activity nor proliferation rate of S2 cells. Complete knock-out of dSETD3 in <i>Drosophila</i> flies did not affect their weight, growth rate or fertility. dSETD3 KO flies showed normal responses to starvation and hypoxia. In sum, we could not identify any clear phenotypes for SETD3 knockout animals, indicating that additional work will be required to elucidate the molecular and physiological function of this highly conserved enzyme.</p></div

Phenotypic characterization of dSETD3 KO flies.

<p><b>(A)</b> Wing size of adult female control (n = 7) and dSETD3 KO (n = 8) flies. Error bars: standard deviation (SD). <b>(B)</b> Weight of control and dSETD3 KO flies (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD). <b>(C)</b> Pupation timing of control and dSETD3 KO animals at 25°C (n = 8 x 30 flies for each condition). Error bars: standard deviation (SD). <b>(D)</b> Fat storage measured by TAG abundance in control and dSETD3 KO flies, normalized to total body weight (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD). <b>(E)</b> Glycogen storage measured in control and dSETD3 KO flies, normalized to total body weight (n = 4 x 8 flies for each condition). Error bars: standard deviation (SD); * <i>t</i> test < 0.05. <b>(F-F’)</b> Full starvation of control and dSETD3 KO animals on PBS/agarose (0.7%) for male (F) or female (F’) adult flies (n = 3 x 20 flies for each condition). Error bars: standard deviation (SD). <b>(G)</b> Fertility of control and dSETD3 KO mated females over the course of three days (n = 4 x 8–10 flies). Error bars: standard deviation (SD); * <i>t</i> test < 0.05. (H) dSETD3 KO flies do not have impaired motility, assayed using a climbing assay with control and dSETD3 KO adult females. Flies were put into plastic tubes, tapped down, and observed climbing towards a light source at the top of the tube. The time was measured that was required for 50% of the flies in one tube to pass a set threshold (biological quadruplicates, each measured twice). Error bars: standard deviation (SD).</p

Generation of dSETD3 KO flies.

<p><b>(A)</b> Schematic of the dSETD3 genomic locus and the knockout region. Orange indicates coding exons. To avoid interfering with the splicing of CG14431 only the first exon of CG32732 (dSETD3) was removed and replaced with a dsRED expression cassette using CRISPR-induced homologous recombination. The location of the amplicon used for Q-RT-PCR in panel C is shown and labeled with ‘qPCR’. <b>(B)</b> Complete loss of dSETD3 protein in different dSETD3 KO lines. Knock-out stock 2 was used in this study. Homozygous flies were lysed and analyzed by immunoblotting with purified dSETD3 antibody. <b>(C)</b> dSETD3 mRNA is completely lost in dSETD3 KO flies. mRNA was isolated from control and dSETD3 KO homozygous flies and dSETD3 expression was analyzed by RT-qPCR against the region indicated in (A), and normalized to rp49. Error bars: standard deviation (SD).</p

dSETD3 is localized in the nucleus and the cytoplasm.

<p><b>(A)</b> Epitope tagged dSETD3 is present in the cytoplasm and the nucleus. S2 cells were transfected with constructs to express either N-terminally or C-terminally HA-tagged dSETD3, and then immunostained for HA-tag or myc-tag (green “FITC”), DAPI (blue) and phalloidin (red “phall”) to stain the actin skeleton. Scale bar: 10μm.</p

Transcriptional effects of dSETD3 KO in adult virgin flies.

<p><b>(A)</b> Control and dSETD3 KO virgin female flies were lysed in TRIZOL, RNA extracted and subjected to microarray analysis. Volcano blot shows all genes that were statistically significantly up or downregulated (<i>t</i> test < 0.05). Genes that were up or downregulated at least 2-fold upon dSETD3 KO (<i>t</i> test < 0.05) are indicated in green (down) and red (up). Data are available under GEO accession number GSE113846. <b>(B)</b> Gene ontology analysis using DAVID of dSETD3-affected genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref041" target="_blank">41</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref042" target="_blank">42</a>].</p

dSETD3 does not play a role in hypoxia response in <i>Drosophila</i>.

<p><b>(A)</b> dSETD3 protein levels do not change upon hypoxia in S2 cells. S2 cells were treated for 5 days with control or dSETD3 dsRNA and then reseeded and subjected to control or hypoxic conditions (1% oxygen) for 36 hours. Cell lysates were analyzed by immunoblotting with indicated antibodies. Quantification of dSETD3 bands normalized to ERK bands was done with ImageJ [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201609#pone.0201609.ref046" target="_blank">46</a>]. <b>(B)</b> Expression of hypoxia induced genes in S2 cells is not dependent on dSETD3. S2 cells were treated for 5 days with control or dSETD3 dsRNA and then reseeded and subjected to control or hypoxic conditions (1% oxygen) for 36 hours. RNA was isolated and analyzed by RT-qPCR. Error bars from technical triplicates: standard deviation (SD). <b>(C)</b> dSETD3 protein levels do not change upon hypoxia in flies. Control flies were subjected to hypoxia (5% oxygen) for 3 or 6 hours and lysates were analyzed by immunoblotting with indicated antibodies. Three samples (5 larvae each) per condition are shown. <b>(D)</b> Expression of hypoxia induced genes is not affected by dSETD3 KO in flies. Control and dSETD3 KO male flies were subjected to 1, 4 or 6 hours of hypoxia (1.8% oxygen). RNA was isolated and analyzed by RT-qPCR. Error bars from technical triplicates: standard deviation (SD). <b>(E-E’)</b> dSETD3 is not required for survival during hypoxic conditions. Control and dSETD3 KO flies were put into hypoxic conditions (1.8% oxygen (E, n = 4 x 10 flies) and 2% oxygen (E’, n = 6 x 10 flies)) for indicated amounts of times and surviving animals after the treatment were counted. Error bars: standard deviation (SD).</p

DENR–MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation

<div><p>The succession of molecular events leading to eukaryotic translation reinitiation—whereby ribosomes terminate translation of a short open reading frame (ORF), resume scanning, and then translate a second ORF on the same mRNA—is not well understood. Density-regulated reinitiation and release factor (DENR) and multiple copies in T-cell lymphoma-1 (MCTS1) are implicated in promoting translation reinitiation both in vitro in translation extracts and in vivo. We present here the crystal structure of MCTS1 bound to a fragment of DENR. Based on this structure, we identify and experimentally validate that DENR residues Glu42, Tyr43, and Tyr46 are important for MCTS1 binding and that MCTS1 residue Phe104 is important for tRNA binding. Mutation of these residues reveals that DENR-MCTS1 dimerization and tRNA binding are both necessary for DENR and MCTS1 to promote translation reinitiation in human cells. These findings thereby link individual residues of DENR and MCTS1 to specific molecular functions of the complex. Since DENR–MCTS1 can bind tRNA in the absence of the ribosome, this suggests the DENR–MCTS1 complex could recruit tRNA to the ribosome during reinitiation analogously to the eukaryotic initiation factor 2 (eIF2) complex in cap-dependent translation.</p></div