Quantitative RT-PCR analysis of PMi for ribosomal protein genes in wild-type and <i>hog1</i> or <i>cbc1</i> mutants.

<p>(A) A decrease in ribosomal protein (RP) pre-mRNAs in response to osmotic stress. The behaviour of transcripts <i>RPL30</i>, <i>RPL28</i>, <i>RPL33A</i>, <i>RPL33B</i>, <i>RPL34A</i> and <i>YDR367W</i> across a time course lasting 5, 10 and 15 minutes after treatment with 0.4 M NaCl was examined by quantitative RT-PCR using specific primers to intron regions and exon regions. The PMi value of the comparison made between stressed (0.4 M NaCl, 15 min) and non-stressed cells per gene, obtained by quantitative RT-PCR, is found in parentheses. Data and error bars represent the average and standard deviation of 3 independent experiments. (B) Representation of PMi for the wild-type, <i>hog1</i> mutant and <i>cbc1</i> mutant cells treated with 0.4 M NaCl for 15 minutes in relation to untreated cells. The intron and exon levels of the transcripts <i>RPL30</i>, <i>RPL28, RPL37A</i>, <i>RPS13</i>, <i>RPL33A</i>, <i>RPL34A</i> and <i>RPL33B</i> were examined by quantitative RT-PCR using specific primers to those regions. Data and errors bars represent the average and standard deviation of 3 independent experiments.</p

Analysis of the relation between ribosomal protein genes transcription and pre-mRNA processing during osmotic stress.

<p>(A) Clustering RP genes according to their transcription rate (TR) profiles. Wild-type cells were grown in YPD until the exponential phase and were then treated with 0.4 M NaCl. The samples taken at 0, 2, 4, 6, 8, 10 and 15 minutes of osmotic shock were processed to measure the TR of all the yeast genes. The data set series for intron-containing RP genes refers to their respective 0 time on a logarithm scale. A relative repression (stress/non-stress ratio, on the log₂ scale) is shown in green (saturated green indicates a decrease of at least 6-fold) and relative induction is depicted red (saturated red indicates an increase of at least 6-fold). RP genes were ordered and grouped into three distinct subclasses by K-means clustering using Euclidean distance. The PMi for all the RP genes are represented in the left panel using a blue-yellow heat map (saturated blue indicates a PMi of ≤−1, while saturated yellow indicates a PMi of ≥1). Grey squares indicate missing values. (B) Comparison of RP genes ΔTR and their PMi in the cells treated with 0.4 M NaCl for 15 minutes related to non-stressed cells. The regression line, equation and correlation coefficient are shown in the graph. The RP genes that have been analyzed by quantitative RT-PCR are indicated as red diamonds.</p

Upf1 and exonuclease Xrn1 contribute to the decrease in ribosomal protein pre-mRNA during osmotic stress.

<p>(A) The total and pre-mRNA relative levels are shown for the non-stressed cells of a wild-type strain and strains with deletions in <i>RRP6</i>, <i>XRN1</i> or <i>UPF1.</i> (B) Representation of PMi in the cells treated with 0.4 M NaCl for 15 minutes in relation to untreated cells for the wild-type strain and the same mutant strains as in (A). The intron and exon levels of the transcripts <i>RPL30</i>, <i>RPL28</i>, <i>RPL37A</i>, <i>RPS13</i>, <i>RPL33A</i>, <i>RPL34A, RPL33B</i> and <i>YDR367W</i> were examined by quantitative RT-PCR using specific primers to those regions. Data and errors bars represent the average and standard deviation of 3 independent experiments.</p

Kinetics of ribosomal protein pre-mRNAs during osmotic stress in cells containing mutations in NMD components.

<p>The behaviour of the transcripts <i>RPL30</i>, <i>RPL28</i>, <i>RPL33A</i>, <i>RPL33B</i>, <i>RPL34A</i> and <i>YDR367W</i> after 0, 5, 10 and 15 minutes treatment with 0.4M NaCl were examined in the wild-type, <i>upf1</i>, <i>upf2</i> and <i>upf3</i> and <i>xrn1</i> mutants by quantitative RT-PCR, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061240#pone-0061240-g002" target="_blank">Figure 2A</a>. Data and errors bars represent the average and standard deviation of 3 independent experiments.</p

Tiling array analysis of exon and intron signals after osmotic stress in relation to non-stress.

<p>(A) Schema of the PM index (PMi) calculation using the intron intensity signal ratio after a 15-minute incubation in the presence of 0.4 M NaCl (stressed cells) in relation to non-stressed cells. The mRNAs without changes, with increases or with a decrease, in the intron signal in relation to the exon signal between the stress and non-stress conditions take values close to zero, or positive or negative PMi values, respectively. All these situations are illustrated with a real example (bottom panels). (B) Global distribution of ICGs (intron-containing genes) according to their PMi (see Material and Methods). The black boxes in the histogram represent the distribution of intron-containing ribosomal protein (RP) genes.</p

Scheme of the changes in ribosomal protein (RP) gene expression upon osmotic stress.

<p>The RP pre-mRNA steady-state depends on both the RP gene transcription rate (TR) by RNA polymerase II and the RP pre-mRNA splicing and degradation rates. The RP mRNA produced by splicing can be used for the synthesis of ribosomal proteins through translation or can be sent to degradation. Osmotic stress provokes a decrease in the RP gene TR and an NMD-dependent increase in the RP pre-mRNA degradation rate. Additionally, osmotic stress increases the RP mRNA degradation rate (lower than the increase in the pre-mRNA degradation rate) and lowers the RP mRNA translation rate. The effect of osmotic stress on RP pre-mRNA splicing and RP decay has not been directly estimated. As a result of these changes, the RP pre-mRNA levels dropped considerably and, to a lesser extent, the RP mRNA level also lowered under osmotic stress. However, RP levels remained constant as a result of the growth inhibition provoked by osmotic stress. See the text for further discussion.</p

Mitochondrial quantitation and network distribution in cultured MNs and sciatic nerves from WT and <i>Gdap1</i>^<i>-/-</i> mice.

<p>Number of mitochondria <b>(A)</b> and network interconnectivity <b>(B)</b> in cultured MNs are represented. The study was performed in the proximal segments (p) and distal segments (d) of WT (black bars) and <i>Gdap1</i>^<i>-/-</i> (gray bars) axons after 24 hour and 48 hour of cell culture. Error bars represent S.E.M. Student’s <i>t</i> test *p<0.05, **p<0.01 and ***p<0.001 <b>(C)</b> Left panel shows semi-thin cross sections of the sciatic nerve from five months old WT and <i>Gdap1</i>^-/- mice. Mitochondria are clearly visible on higher magnification images of transversal section (right panel). Mitochondrial axonal content was quantified by electron microscopy on proximal and distal cross sections of the sciatic nerve. (n = 4; Error bars represent S.E.M.; asterisks indicate significant differences between WT and <i>Gdap1</i>^<i>-/-</i> mice, Mann-Whitney test, **p<0.01,***p<0.001). <b>(D)</b> Measurement of mitochondrial DNA (mtDNA) copy number in sciatic nerves.</p

Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.

<p>Mitochondrial shape descriptors were measured in 20 WT and 30 <i>Gdap1</i>^-/- motorneurons. Student’s t test was performed for normal distributed parameters (number of mitochondria, circularity, roundness and aspect ratio) and Mann-Whitney U test for those that were non-normal distributed (surface area, Feret´s diameter and perimeter). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005115#pgen.1005115.g006" target="_blank">Fig 6</a> for a visual representation.</p><p>*p<0.05,</p><p>**p<0.01.</p><p>Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.</p

Postranscriptional modification of the tubulin cytoskeleton in primary sensory and motor neuron cultures.

<p><b>(A)</b> DRG sensory neurons and <b>(B)</b> embryonic MNs were double-stained for acetylated α-tubulin (acetylated α-tub, green) and β-III tubulin (β-III tub, red). As indicated by respective histograms there is a significant reduction of acetylated α-tubulin in both MN and sensory neurites in <i>Gdap1</i>^<i>-/-</i> mice. Graph represents means and S.E.M of 3 independent culture preparation per genotype. Student’s <i>t</i> test ***p<0.001.</p

Behavioural testing and electrophysiological measurements on <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Upper panel shows photographs of 3 months-old mice suspended by its tail. WT mice show a characteristic response trying to escape by splaying its hind limbs away from the trunk of its body. In contrast, hind limbs of <i>Gdap1</i>^-/- mice are held tonically against its trunk in an abnormal dystonic posture. Lower panels display a low body position and a dragging tail present in <i>Gdap1</i>^<i>-/-</i> mice as compared to age-matched WT mice. <b>(B)</b> Motor coordination was assessed by rotarod test, (n = 10 for each genotype and at each age group). <b>(C)</b> Representative hind limb walking patterns of 5 months-old WT and <i>Gdap1</i>^-/- mice where the stride length (SL) and stride angle (SA) have been depicted. Footprints revealed that <i>Gdap1</i>^<i>-/-</i> mice walk with an abnormal gait. The scheme of a hindpaw footprint indicating measured parameters (PL: plantar length; TS: toe spreading) has been included. <b>(D)</b> Quantification of various parameters obtained from the gait analysis of WT (black columns) and <i>Gdap1</i>^<i>-/-</i> (grey columns) animals at 5 and 12 months of age. Upper graphs show stride length (left) and stride angle (right). Lower graphs show the quantitative analysis of the hindpaw footprint parameters toe spreading (left) and plantar length (right). Analysis was conducted on 10 clearly visible footprints at 5 animals per genotype. Determination of sciatic nerve compound muscle action potential (CMAP) amplitudes at both distal and proximal <b>(E)</b> as well as motor nerve conduction velocities (MNCV) <b>(F)</b> measured in WT and <i>Gdap1</i>^-/- mice at 2 and 5 months of age (n = 4). Error bars indicate standard error of the mean (S.E.M.). <i>p</i> values were calculated using Student's <i>t</i> test,*p<0.05, **p<0.001, ***p<0.0001.</p