<i>rpoa-2(op259)</i> mutants have altered ribosome synthesis.

<p>A) Most cells in <i>C. elegans</i> exhibit a single but often very prominent nucleolus. In germ cells of the meiotic pachytene stage, the nucleoli (inner white dashed circle) impose as central spheres of approximately half the diameter of the containing nuclei (outer white dashed circle). In <i>rpoa-2(op259)</i>, the nucleolar diameter is reduced. Many of the germ cell nucleoli show a substructure by DIC that is enlarged in <i>rpoa-2(op259)</i> mutants (black arrows). Size bar, 10 µm. B) Nucleolar volume calculated from measures of the diameter in DIC images of the late meiotic pachytene region of wild-type, <i>hus-1(op241)</i>, <i>cep-1(gk138)</i>, <i>ced-3(n717)</i>, <i>rpoa-2(op259)</i>, <i>ncl-1(e1865)</i>, and <i>rpoa-2(op259); ncl-1(e1865)</i> gonads. Error bars, SD of nucleolar size over all animals tested (12 animals, 5 nucleoli per animal). C) Quantification of ribosomal RNA in whole worm extracts by qRT-PCR. The mean levels of the mature rRNAs (competimer strategy) and of transcribed spacer sequences of the pre-rRNA were normalised by the internal controls in each sample (Pol II transcripts) and compared to N2 wild type. At least 9 or 4 samples per strain were probed for rRNAs or pre-rRNAs, respectively; error bars, 95% CI of the mean. D) Ribosomal proteins represent a smaller fraction of total proteins in <i>rpoa-2(op259)</i> mutants compared to wild-type animals. Individual protein abundance was determined by spectral counting and a quantitative value was assigned by the Scaffold 3 software <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943-Searle1" target="_blank">[109]</a>. Ribosomal proteins were grouped as small or large ribosomal subunit proteins, or a small group of acidic ribosomal proteins. A subset of structural proteins is included as a control group. Similar numbers of proteins were identified in each group for both samples (numbers inside circle). Mean ratio of protein abundance in <i>rpoa-2(op259)</i> over wild type is indicated. E) Ethidium bromide-stained denaturing agarose gel with 800 ng total RNA from adult wild-type and <i>rpoa-2(op259)</i> mutant worms. An approximately 2.8 kb distinct band running between the 26S and 18S ribosomal RNAs is more prominent in <i>rpoa-2(op259)</i> (arrow). Characterisation of its sequence and its endings (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943.s023" target="_blank">Text S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003943#pgen.1003943.s011" target="_blank">Fig. S11</a>) revealed a precise truncation of the mature 26S rRNA and no addition of a poly-A tail. The base numbers indicate the position in the reference sequence of the rDNA repeat unit (top) or along the 26S rRNA (bottom), respectively. F) Relative abundance of the 26S-short rRNA in <i>rpoa-2(op259)</i>, rRNA processing mutants (<i>pro-2</i>, <i>pro-3</i>), translation initiation factor mutants (<i>ife-1</i>, <i>ife-2</i>), apoptosis mutants (<i>cep-1</i>, <i>ced-3</i>), an exosome mutant (<i>crn-3</i>), and of double mutant combinations with <i>rpoa-2(op259)</i>. On each northern blot membrane, the ratio of the 26S-short to 26S rRNA signals was arbitrarily set to 1.0 for the wild-type controls. Mean relative ratios, 95% CI of the mean, and number of samples analysed per strain.