Targeted mutations in dEndoA-BAR and their relationship to the structure of hEndoA1-BAR.

<p>A, Schematic representation of the mutations introduced in the rescue constructs encoding dEndoA-BAR. B, Mutations homologous to the mutations in dEndoA-BAR (A), mapped onto the tertiary structure of hEndoA1-BAR monomer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009492#pone.0009492-Masuda1" target="_blank">[PDB code 1X03A, 8]</a>. The central helix-loop appendage (<i>red</i>) and the residues constituting the hydrophobic ridge (<i>yellow</i>) are indicated. The residues mutated to change the BAR domain curvature are also indicated (<i>pink</i>), as are the three electropositive lysine residues that were mutated to electronegative glutamic acid residues (<i>light green</i>). The <i>inset</i> at the lower right shows the BAR dimer, with the two monomers colored <i>gray</i> and <i>blue</i>. C, Primary structure alignment of hEndoA1-BAR (accession BAE44459.1; <i>top</i>) and dEndoA-BAR (accession CAD24682.1; <i>bottom</i>). The alpha-helical secondary structure is indicated by <i>squiggles</i>, based on the hEndoA1-BAR structure. The residues associated with the hydrophobic ridge are also indicated (<i>closed triangles</i>).</p

The small-eye trait induced by expression of the <i>UAS-endoA^A66W</i> mutant transgene.

<p>A, eye of a control fly carrying the <i>elav-GAL4</i> driver but no <i>endoA</i> transgene. B, <i>endoA</i> null fly rescued to the pharate adult stage by <i>UAS-endoA^A66W</i> expression, driven by <i>elav-GAL4</i> (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA^Δ4/endoA^Δ4</i>). Note that the eye size is reduced and that the lower eye tip is pointy rather than rounded. C, The small-eye trait also appears when <i>UAS-endoA^A66W</i> expression occurs on a wild-type <i>endoA</i> background (<i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i>). D–G, Scanning electron micrographs of <i>elav-GAL4</i> (D, F) and <i>elav-GAL4/w</i>; <i>UAS-endoA^{A66W 4.1}/+</i>; <i>endoA⁺/endoA⁺</i> (E, G) eyes. In G, some examples of ommatidia that lack bristles are indicated by <i>asterisks</i>, and aberrant dual bristles by <i>arrowheads</i>. Pitting is indicated by an <i>arrow</i>. Scale bars: C, 100 µm (applies to A–C); E, 50 µm (D, E); G, 20 µm (F, G).</p

Life span and locomotor activity of adult rescuants.

<p>A, Kaplan-Meier survival curves showing the post-eclosion life span of the rescuants. The <i>UAS-endoA</i>* transgene either carried the mutations indicated in the Figure, or wild type <i>endoA</i> (“wt”). B, survival curves for the <i>EndoA(Arf)-HA</i> rescuants (<i>closed squares</i>) and control rescuants carrying an HA-tagged wild type <i>endoA</i> transgene (“wt-HA”, <i>open squares</i>). C, The median survival time (MST) of individual transgenic integration lines. On average, 32 flies per line were included in the survival analysis (range 12–52). D, The locomotor activity period (LAP; mean ± SEM). On average, 27 flies per line were included in the locomotion analysis (range 12–37). For statistical analysis, the rescue of EndoA(Arf)-HA was compared with the rescue provided by the HA-tagged <i>endoA⁺</i> transgene (“wt-HA”). Otherwise, the rescuants associated with the untagged <i>endoA⁺</i> transgene were used as controls (“wt”). ^*<i>P</i><0.01, ^**<i>P</i><10⁻⁹. N.s., not significant.</p

Neurotransmission at the neuromuscular junction in larval mutant rescuants.

<p>Intracellular recordings were made from the somatic muscles of <i>Elav-GAL4/Y or w</i>; <i>UAS-endoA</i>*<i>/+</i>; <i>endoA^Δ4/endoA^Δ4</i> third instar larvae, where <i>UAS-endoA</i>* represents a mutated <i>endoA</i> transgene, or one encoding wild type EndoA (“wt”), as indicated in A–F. The suffix “−HA” signifies the presence of an additional HA tag. Raw recordings are not shown. A, Ability to sustain neurotransmitter release during a tetanus (10 min at 10 Hz) and immediately following tetanic stimulation (10 min at 0.2 Hz). The amplitude of the excitatory junctional potential (EJP), relative to the amplitude prior to the tetanus (0.2 Hz, not shown), is plotted. Error bars are omitted in A for clarity; the variability can be judged from B–D. B, The EJP amplitude (mean and 95% confidence interval) at the end of the 10 Hz tetanus, just before switching back to stimulation at 0.2 Hz (<i>arrow</i> in A). C, The maximal EJP amplitude (mean and 95% confidence interval) observed in the 10 min post-tetanic recovery period. D, The proportion of cases, in which the EJP took less than two minutes to recover from the end-tetanic level (arrow in A) to at least 75% of the maximal post-tetanic EJP amplitude, after switching from 10 Hz to 0.2 Hz stimulation. The bars indicate 95% confidence intervals. E and F, Frequency and amplitude of miniature excitatory junctional potentials (mEJPs). For each genotype and line, <i>n</i> is shown above the bars. N.s., not significant.</p

Expression capability of <i>UAS-endoA</i>* transgenes.

<p>A, Western blots of extracts from late-stage embryos, probed simultaneously with anti-EndoA and anti-Elav primary antibodies. Shown are genotypes without transgenes (<i>w¹¹¹⁸</i> and <i>elav-GAL4</i>), <i>EndoA^Δ4</i> null mutants, and null mutants carrying the indicated <i>endoA</i> transgenes driven by <i>elav-GAL4</i>. Note that some genotypes appear more than once. The EndoA immunosignal (wild type or mutant) generally runs as a doublet with the lower band matching the predicted size of EndoA (41.4 kDa). The signals from Elav and an unidentified protein (<i>asterisk</i>) both serve as loading controls. The <i>UAS-endoA^{EndoA(Arf)-HA}</i> product runs distinctly lower than other products, due to the deletion of the entire BAR appendage. B, Extracts from null mutants carrying the indicated HA-tagged <i>endoA</i> transgenes driven by <i>elav-GAL4</i>, probed simultaneously with anti-HA and anti-Elav. C, Extracts from fly heads of EndoA nulls, rescued to adulthood with mutant EndoA transgenes (“successful transgenes”) and probed with anti-EndoA and anti-Elav. Numbers above lanes in all panels are for reference only.</p

Ability of transgenic <i>endoA</i> constructs to rescue the development of <i>endoA</i> null mutants to adulthood.

<p>Shown is the proportion of eclosed adult rescuants (genotype <i>elav-GAL4/Y or w</i>; <i>UAS-endoA</i>*<i>/+; endoA^Δ4/endoA^Δ4</i>) relative to the total number of adult progeny resulting from the rescue cross. The <i>UAS-endoA</i>* transgene carried the mutations indicated on the abscissa and in some cases also encoded a hemagglutinin epitope tag (indicated by the suffix “−HA”). Also shown is the proportion of rescuants in which the <i>endoA</i> transgene encoded either wild type EndoA (“wt”), or HA-tagged wild type EndoA (“wt-HA”). Each <i>bar</i> represents one transgenic integration line, specified <i>below the abscissa</i>. The total number of adult progeny resulting from the rescue cross is indicated for each line (<i>numbers above the bars</i>). The lower and upper 95% confidence intervals are given. ^*<i>P</i><0.01. N.s., not significant. ^†Besides <i>UAS-endoA^{A66W 4.1}</i>, the rescue efficiency of two other <i>UAS-endoA^A66W</i> transgenes was evaluated (<i>UAS-endoA^{A66W 41.3}</i> and <i>UAS-endoA^89.1</i>). They both caused lethality of all the progeny from the rescue cross, as detailed in the text.</p

<i>In vitro</i> protein-RNA UV crosslinking analysis of <i>ct</i>Utp4.

<p><b>(A)</b> Hits obtained from deep sequencing analysis of <i>Chaetomium thermophilum</i> His₆-Utp4 (coverage, blue) mapped within the 5'-ETS (nucleotides 1 to 587) after UTP-A/5'-ETS RNP assembly by co-expression in yeast. <b>(B)</b> Mutations (deletions and substitutions) identified after cDNA library synthesis are indicated by red bars. Mutational hot spots observed in the two crosslinked regions are labeled accordingly (G66 and A220). As background control, the UTP-A/5'-ETS complex carrying untagged Utp4 (“no His₆ tag”) was used. The crosslinked region around 5'-ETS bases 100–140, which was found also in the untagged control, is marked with an asterisk. <b>(C)</b> and <b>(D)</b> The two main regions of the 5'-ETS (A53-C96 and A192-C235) that were crosslinked to His₆-Utp4 are shown together the number of mutations per base. The respective 5'-ETS sequence is depicted below. Mutational hot spots G66 and A220 colored in red.</p

The UTP-A complex from <i>Chaetomium thermophilum</i>.

<p><b>(A)</b> Scheme showing the spatial assembly of the fungal UTP-A complex including the Utp4 X-ray structure and the EM-modeled 5'-ETS. The propellers of remaining UTP-A proteins (Utp8, Utp15, and 2× Utp17) are placed according previous biochemical and EM-studies. The α-solenoidal parts (including whole Utp5) are not included. The entire Utp10 molecule turning around Utp4 is interpreted as also the very C-terminus (atomic model) of Utp8 next to the Velcro-closure of Utp4. The position of the disease-modified arginine in human Utp4 in the interface to Utp10 is highlighted within a red sphere. <b>(B)</b> Comparison of the UTP-A complexes from <i>Chaetomium thermophilum</i> (left panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>]) and <i>Saccharomyces cerevisiae</i> (right panel; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref028" target="_blank">28</a>]). While the overall architecture is conserved, the 5'-end of the RNA shows a different arrangement. In addition, the Upt8-Utp4 contact is not visible in the yeast structures.</p

Model of co-transcriptional assembly of the 90S pre-ribosome.

<p>The nascent 5'-ETS (black line) recruits the early 90S modules (UTP-A, UTP-B, and U3 snoRNP) in a hierarchical fashion, with the UTP-A complex being the first one that binds to the extreme 5'-end of the pre-rRNA. This early assembly intermediate, together with the subsequently transcribed pre-18S rRNA (yellow line) and additional factors, forms the 90S pre-ribosome. Complexes are labeled accordingly. The 3'-hinge region is highlighted in pink. Figure is adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178752#pone.0178752.ref009" target="_blank">9</a>].</p

Uncommon Velcro-closure of the C-terminal β-propeller 2.

<p><b>(A)</b> Schematic representation of the last blade 14 of Utp4. The four β-strands of the blade are represented as arrows in different colours: 14A and B (C-terminus of β-propeller 2, red), 14C (N-terminus of β-propeller 1, blue), and 14D ((His)₆-TEV-tag, grey). <b>(B)</b> Close-up of blade 14 complemented by the very N-terminus of the polypeptide chain, forming an uncommon parallel β-strand 14C (blue) and the artificial TEV site (grey) forming an antiparallel β-strand 14D. The highly conserved residues and their hydrogen-bonding network stabilizing the blade and therefore the Velcro-closure of β-propeller 2 are represented in sticks. Salt-bridges are indicated by dashed lines.</p