Compromising PARP1 enzymatic activity disrupts co-localization of nucleolar proteins.

<p>A–D. The dissected pairs of salivary glands expressing CC01311 nucleolar GFP were split into left and right individual glands and stained separately using antibody against nucleolar protein Fibrillarin (A, C) and nucleolar antibody AJ1 (B, D). Three nucleolar markers, Fibrillarin, CC01311, and AJ1 co-localize in wild-type third-instar larvae salivary gland nucleoli <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002442#pgen.1002442-The1" target="_blank">[53]</a>, but show completely different localization in <i>Parp^RNAi</i>-expressing tissues (C–D). Nuclear membrane envelop is outlined. E–F. Chemical inhibition of PARP1 leads to immediate disruption of nucleolar domain. Wild-type third-instar larvae (E) were cultured 12 hours in the presence of a NAD analogue PARP1 inhibitor, 3-aminobenzamide (F). Nucleoli were detected by anti-Fibrillarin antibody (green). DNA was detected by Draq5 dye (red). The separation of a single nucleolar domain (E) into multiple “blobs” is clearly seen upon PARP1 inhibition (F). G. Prolonged treatment of third instar larvae with PARP1 inhibitor. Nucleoli were detected by anti-Fibrillarin antibody (green). PARP1 was detected by PARP1-DsRed autofluorescence (red). Arrows indicate nucleoli-like blobs.</p

Production of rRNA intermediates increases upon disruption of PARP1 or PARG activity.

<p>A. Diagrammatic representation of mammalian rRNA transcript processing. The final 18S rRNA product becomes part of the 40S small ribosomal complex, while the 5.8S and 28S rRNA transcripts are incorporated into the large 60S ribosomal complex. Red bar above the scheme indicates internal transcriber spacer (ITS) probe, which was used to detect intermediates of rRNA processing on Northern blots. B. Northern blot analysis of rRNA intermediates. Disruption of PARG or PARP1 activity enhances the production of rRNA intermediates (right lanes) compared to the heterozygous (left lanes), which has normal PARP1 and PARG activity. The total level of mature rRNA does not increase (18S and 28S). Labeled probe to <i>Drosophila</i> Tubulin mRNA was used as a loading control.</p

Nucleolar proteins show differential interaction with pADPr.

<p>Immunoprecipitation assays using mouse anti-pADPr antibody. Wild-type (A) and <i>Parg^27.1</i> mutant (B) third-instar larvae were used. The following antibodies were used for Western blot analysis: rabbit anti-Fibrillarin; rabbit AJ1; rabbit anti-Nucleolin; rabbit anti-GFP (to detect nucleolar CC01311 marker); rabbit anti-Nucleophosmin; rabbit anti-dNop5; rabbit anti-CK2α; and mouse anti-Dlg (as a control).</p

PARP1 is required for ribosomal biogenesis.

<p>A–D. TEM images of sections through midintestine of wild-type (A, C) and <i>Parp^CH1</i> mutant second-instar larvae (B, D). Sections were made through the regions indicated with arrows in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002442#pgen-1002442-g001" target="_blank">Figure 1C and 1D</a>. Rectangles outline areas magnified in panels C and D. Although concentration of ribosomes seems to be identical in <i>WT</i> and <i>Parp^CH1</i> mutant, the total volume of cytoplasm is much smaller in <i>Parp^CH1</i>. White bar shows cell size difference between <i>WT</i> and <i>Parp^CH</i>. E–F. Sucrose density gradient analysis reveals the difference between ribosomal profiles in wild-type (<i>WT</i>), <i>Parg^27.1</i> and <i>Parp^C03256</i> mutants. E. A260 profiles of ribosome pools separated over sucrose density gradients. Positions of fractions corresponding to 40S and 60S subunits, 80S ribosomes and polysomes are indicated. F. Total proteins were extracted from corresponding fractions after sucrose density gradients (which are shown on panel E) and subjected to Western blot analysis using antibody against RPS6 protein, which belongs to the 40S ribosomal subunit. In wild-type samples, RPS6 protein labels fraction 15 (40S subunit itself), fractions 7–11 (mono-ribosome) and fractions 1–5 (polysomes). No polysomes were detected in either <i>Parg^27.1</i> or <i>Parp^C03256</i> mutants. Total level of mature mono-ribosomes is significantly decreased in <i>Parg^27.1</i> mutants, although, total level of RNA (E, fractions 13–19) is much higher than in <i>WT</i>. Although antibody against ribosomal protein reveals ribosomal-related particles in fractions 7–15 in <i>Parp^C03256</i> mutant, those particles could not be separated by sucrose gradient (E shows no picks). Last observation suggests incomplete processing or misfolding of ribosomes in <i>Parp^C03256</i>. G. Detection of mRNA in Polysome Fractions. Quantitative real-time RT-PCR was used to analyze the amount of mRNA translated after disrupting PARP1 activity to measure functional ribosome complex formation. mRNA was isolated from 3rd instar larvae before and after polysome fractionation. Polysome fractions from each sample were combined together after isolating mRNA. Each dataset was normalized using Tubulin. The chart shows values obtained after normalizing each value generated before fractionation to values after polysome fractionation. Bars on the chart represent two independent experiments.</p

PARP1 controls nucleolar structural integrity.

<p>A–B. PARP1 protein localizes to nucleoli in all <i>Drosophila</i> tissues, including polytene nuclei of larval salivary glands (A) and diploid nuclei of larval brain (B). The dissected larval salivary glands and brains expressing PARP1-DsRed (red) were stained with the DNA binding dye Draq5 (green). Positions of nucleoli are indicated with arrows. SG – larval salivary gland; BR – larval brain. C–D. PARP1 deletion displaces nucleoli protein as detected by nucleoli-specific antibody AJ1 (red). AJ1 detects nucleoli in every cell of <i>Drosophila</i> midintestine in wild-type second-instar larvae (C), but only in a few cells in <i>Parp^CH1</i> mutants (D). DNA is detected with OliGreen dye (green). E–H. Deletion or disruption of PARP1 protein functions disintegrates nucleolus structure. Salivary glands from wild-type (E), <i>Parp^RNAi</i> expressing (F), hypomorphic <i>Parp</i> mutant, <i>Parp^C03256</i> (G), and overexpressing antagonist of PARP1, PARG (H) 3rd instar larvae were stained for the nucleolar specific protein Fibrillarin (red). In wild-type cells, Fibrillarin (red) localizes in an intact single nucleolus (E). Compromising PARP1 protein activity (F–H) causes nucleolus fragmentation, as indicated by the aberrant localization of Fibrillarin (red). DNA is detected with Draq5 dye (green) (E–G). Overexpression of PARG-EGFP protein (H) is detected by EGFP autofluorescence (green). Arrow (F) indicates the nucleus of <i>Parp^RNAi</i>-expressing larval salivary gland that shows undetectable levels of Fibrillarin protein. White bars of (F) indicate areas that were subjected to TEM analysis shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002442#pgen-1002442-g006" target="_blank">Figure 6A–6D</a>. I–K. Nucleolar fragments in <i>Parp^C03256</i> and <i>Parg^27.1</i> do not contain rDNA. Dissected salivary glands from wild-type (I) <i>Parp^C03256</i> (J) and <i>Parg^27.1</i> (K) 3rd instar larvae were hybridized with rDNA probe (red) and stained for the Fibrillarin protein (green). DNA was detected with Draq5 dye (blue).</p

Nuclear PARP1 facilitates ribosomal biogenesis: a model.

<p>PARP1 protein becomes automodified upon each act of transcriptional start within rDNA gene and serves as a chaperoning machine during whole cycle of ribosome maturation in nucleolus. The dynamic Poly(ADP-ribose) tree forms a network, which organizes specific nucleolar microenvironment, brings a subset of nucleolar protein (such as Fibrillarin and AJ1) to the proximity of precursor rRNA, and coordinates the order of events of rRNA processing, modification, and loading of subsets of ribosomal proteins. Depletion of PARP1 protein leads to removal of pADPr-binding proteins from nucleoli, which disrupts processing, modification and folding of ribosomal RNA. PARG protein is required to 1) restart the system and 2) recycle protein components after completion of one cycle of ribosomal subunit synthesis.</p

Mutating PARG disrupts nucleoli and reveals differential localization of nucleolar proteins.

<p>A–B. The structure of wild-type nucleolus detected by EM microscopy (A) is affected in <i>Parg^27.1</i> mutants (B). In contrast to clear homogeneous (grey) content of wild-type nucleolus, <i>Parg^27.1</i> mutant nucleoli accumulate “holes” and “aggregates” of proteins or chromatin (black). Arrows indicate the nucleolus. Arrowheads are pointing to the nuclear envelope. C. Nucleolar proteins colocalize with PARP1 (red) in wild-type nucleoli. Fibrillarin (green) protein is shown. D–G. Mutating PARG displaces PARP1 protein from chromatin to Cajal Bodies (arrowheads) and “traps” PARP1 within condensed nucleolar blocks (arrows). One class of nucleolar proteins completely co-localizes with PARP1 in CB and nucleoli (D–E). Another group of nucleolar proteins (F–G) behave independently from PARP1. This group of proteins has a homogeneous localization in nucleoli (arrows) and could barely be detected in CBs (arrowheads).</p