PARP1 is required for ribosomal biogenesis.
A–D. TEM images of sections through midintestine of wild-type (A, C) and ParpCH1 mutant second-instar larvae (B, D). Sections were made through the regions indicated with arrows in Figure 1C and 1D. Rectangles outline areas magnified in panels C and D. Although concentration of ribosomes seems to be identical in WT and ParpCH1 mutant, the total volume of cytoplasm is much smaller in ParpCH1. White bar shows cell size difference between WT and ParpCH. E–F. Sucrose density gradient analysis reveals the difference between ribosomal profiles in wild-type (WT), Parg27.1 and ParpC03256 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 Parg27.1 or ParpC03256 mutants. Total level of mature mono-ribosomes is significantly decreased in Parg27.1 mutants, although, total level of RNA (E, fractions 13–19) is much higher than in WT. Although antibody against ribosomal protein reveals ribosomal-related particles in fractions 7–15 in ParpC03256 mutant, those particles could not be separated by sucrose gradient (E shows no picks). Last observation suggests incomplete processing or misfolding of ribosomes in ParpC03256. 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.
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