Calcium induced fragmentation of small rRNAs in extracts of cells expressing MNase fusion proteins of rpL5 or rpL35.

<p>Cellular extracts of yeast strains expressing no MNAse fusion protein (Y206) or MNAse fusion proteins of rpL35 (Y2371) or rpL5 (Y2369) were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Samples were taken before addition of calcium chloride (0 minutes) or after extract incubation in the presence of calcium chloride for the indicated times at room temperature (22°C). Total RNA was extracted and separated by size on denaturing polyalcrylamide gels as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Gels were stained with ethidium bromide (A) or were further processed for Northern blotting (B–E) and probed with oligonucleotides detecting the 5S rRNA ((B), oligonucleotide O2474), the 5′ end of 5.8S rRNA ((C), oligonucleotide O209), the 3′ end of 5.8S rRNA ((D), oligonucleotide O2959) or the 25S rRNA region 1623–1643 nucleotides downstream of the 5′ end of 25S rRNA ((E), oligonucleotide O3068). ). Positions of 5.8S rRNAs, 5S rRNA and tRNAs are indicated. Stars (*) indicate positions of strain specific rRNA fragments generated during the course of extract incubation in the presence of calcium chloride. A cross (+) marks a RNA fragment significantly detected in extracts of strains not expressing MNase fusion proteins.</p

Calcium induced fragmentation of large rRNAs in extracts of cells expressing MNase fusion proteins of rpL5, rpL35 or rpS13.

<p>Cellular extracts of yeast strains expressing no MNAse fusion protein (Y206) or MNAse fusion proteins of rpL35 (Y2371), rpL5 (Y2369) or rpS13 (Y2361) were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Samples were taken before addition of calcium chloride (0 minutes) or after incubation in the presence of calcium chloride for the indicated times at room temperature (22°C). Total RNA was extracted and separated by size on agarose gels as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Gels were stained with ethidium bromide (A) or were further processed for Northern blotting (B–D) and probed with oligonucleotides detecting the 5′ end of 25S rRNA ((B), oligonucleotide O1821), the 3′ end of 25S rRNA ((C), oligonucleotide O1896) or the 3′ end of 18S rRNA ((D), oligonucleotide O1957). Positions of 25S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNAs are indicated. Stars (*) indicate positions of strain specific rRNA fragments generated during the course of extract incubation in the presence of calcium chloride.</p

Location of rpL5-MNase cleavages in 25S-preLSU and 7S-preLSU rRNA tertiary structure models.

<p>Structures of rRNA, rpL5, rpL27 and rpL35 in 25S-preLSUs (A) or 7S-preLSUs (B) are shown using the color scheme as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.g001" target="_blank">Fig 1</a>, with rRNA cleavage sites at the surface highlighted in yellow. The orientation shown is centered on the central protuberance. The pdb files 5apo and 3jct used to create the Figure were published in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref016" target="_blank">16</a>].</p

Changes in r-protein assembly states of early LSU precursors after <i>in vivo</i> depletion of LSU r-proteins required for early LSU pre-rRNA processing.

<p>The dataset generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g003" target="_blank">Fig 3</a> was analyzed in respect to changes in r-protein levels in Noc2-TAP fractions isolated from wild type cells or from r-protein expression mutants. Observed changes in levels of r-proteins and the results of clustering analyses are visualized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g003" target="_blank">Fig 3B</a>. Groups of r-proteins mentioned in the text are highlighted by bars on the right. Only r-proteins which were identified in at least 70% of the 17 pairwise comparisons were included in these analyses. The r-proteins whose expression was shut down in the respective experiment (“mutant <i>versus</i> wild type”) are highlighted by a red box.</p

Interactions of dII/dVI cluster r-proteins with rRNA in mature ribosomes.

<p>The yeast LSU is shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g001" target="_blank">Fig 1</a> viewed from the solvent exposed side in <b>A)</b>, <b>C)</b> and <b>E)</b>. It is rotated by approximately 90 degree around a horizontal axis in <b>B)</b>, <b>D)</b> and <b>F)</b>. In <b>A)</b>–<b>F)</b> LSU rRNA domain II is colored in yellow, LSU rRNA expansion segment 7 in orange, LSU rRNA domain VI in blue, 5.8S rRNA in red and other parts of the LSU rRNA in grey. In <b>A)</b> and <b>B)</b> dII/dVI cluster r-proteins are the only r-proteins shown and colored in shades of green. In <b>C)</b> and <b>D)</b> only rRNA is visualized and in <b>E)</b> and <b>F)</b> rpL16/uL13 is the only r-protein shown, with its globular domain in light green and its C-terminal clamp-like domain in dark green.</p

Influence of the linker size of MNase-rpL35 fusion proteins on rRNA fragmentation.

<p>Cellular extracts of yeast strains expressing MNAse fusion proteins of rpL35 in which the MNase and the rpL35 coding regions are separated by 2 (Y2510, lanes 1–4), 41 (Y2511, lanes 5–8) or 24 (Y2512, lanes 9–12) amino acids were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Samples were taken before addition of calcium chloride (0 minutes) or after incubation in the presence of calcium chloride for the indicated times at room temperature (22°C). Total RNA was extracted and separated by size on agarose gels (A) or on polyalcrylamide gels (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#s4" target="_blank">Materials and Methods</a>. Gels were stained with ethidium bromide. Positions of 25S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNAs are indicated. Stars (*) indicate positions of strain specific rRNA fragments generated during the course of extract incubation in the presence of calcium chloride. Two stars (**) indicate positions of rRNA fragments appearing in higher amounts in the strain expressing MNAse linked to rpL35 by 41 amino acids. A cross (+) marks a RNA fragment significantly detected during the time course of incubation in strains not expressing MNase fusion proteins.</p

Changes in the association of LSU biogenesis factors with early LSU precursors after <i>in vivo</i> depletion of LSU r-proteins required for early LSU pre-rRNA processing.

<p>Conditional r-protein expression mutants or wild type cells were cultivated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#pone.0143768.g002" target="_blank">Fig 2</a>. Noc2-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#sec008" target="_blank">Materials and Methods</a>). Proteins in Noc2-TAP fractions were identified and quantified by mass spectrometry. Isobaric labeling of peptides (iTRAQ, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#sec008" target="_blank">Materials and Methods</a>) was used to directly compare levels of individual proteins in Noc2-TAP fractions from wild type cells with the respective levels in Noc2-TAP fractions from conditional r-protein expression mutants. In <b>A)</b> is shown the average proteome composition of nine Noc2-TAP fractions analyzed in this study by mass spectrometry. In <b>B)</b> the changes in levels of individual LSU biogenesis factors in Noc2-TAP fractions from mutant versus wild type cells as determined by iTRAQ analyzes are summarized. Each pair wise comparison (“mutant <i>versus</i> wild type”) was performed at least twice starting from independent cell cultures. Data from 17 pairwise comparisons were used to detect co-behaving groups of LSU biogenesis factors by a clustering algorithm (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143768#sec008" target="_blank">Materials and Methods</a>). Similar behaving proteins are grouped in the same branch of the dendrogram depicted on the left. The iTRAQ ratios of LSU biogenesis factors for each individual pairwise comparison are shown as heat map using the color code depicted on the upper right. Only LSU biogenesis factors were included in these analyses, which were identified in at least 70% of the 17 pairwise comparisons. Groups of LSU biogenesis factors mentioned in the text are highlighted by bars on the right.</p

Analysis of major calcium induced rRNA 5′ ends in MNase-rpL5 or MNase-rpL35 containing ribosomes.

<p>Cellular extracts of yeast strains expressing no MNase fusion protein protein (Y206) or MNAse fusion proteins of rpL35 (Y2371) or rpL5 (Y2369) were incubated for 50 minutes at room temperature in the presence of calcium chloride. Total RNA was purified and analyzed by primer extension with primers hybridizing to a region 1623 nucleotides downstream of the 25S rRNA 5′ end ((A), oligonucleotide O3068)), a region 1855 nucleotides downstream of the 25S rRNA 5′ end ((B), oligonucleotide O1890), a region 143 nucleotides downstream of the 5.8S rRNA 5′ end ((C), oligonucleotide O2959) or a region 1068 nucleotides downstream of the 25S rRNA 5′ end ((D), oligonucleotide O3074). Primer extension products were separated by size by denaturing acrylamide gel electrophoresis and analyzed by autoradiography. Sequencing reactions of a plasmid carrying a full ribosomal DNA copy (K375) were performed in parallel (lanes 4–6 in A–D). Major detected 5′ ends are indicated on the left and were named rpL35-C1, rpL35-C2, rpL35-C3, rpL5-C1 and rpL5-C2. On the right in figures A–D secondary structure diagrams adapted from <a href="http://www.rna.ccbb.utexas.edu/" target="_blank">http://www.rna.ccbb.utexas.edu/</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#pone.0042449-Cannone1" target="_blank">[15]</a> of the rRNA regions of interest are shown with major detected 5′ ends highlighted in red. Helix and expansion segment numbering is according to the <i>E. coli</i> 23S rRNA helix numbering and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042449#pone.0042449-BenShem1" target="_blank">[11]</a>.</p

Location of tethered MNase cleavages in secondary and tertiary structure models of the ITS2 rRNA region.

<p>Structures of rRNA, rpL5, rpL27 and rpL35 in 7S-preLSUs are shown using the same color scheme as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.g001" target="_blank">Fig 1</a>, centering on LSU rRNA domains I and III in (A) and on the subunit interface side in (B). The last nucleotides of the terminal ITS2 region resolved in the 7S-preLSU structure model are colored in yellow. The pdb file 3jct used to create the figures (A) and (B) was published in ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref016" target="_blank">16</a>]). In (C-E) three secondary structure models of the ITS2 region are shown which were taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref029" target="_blank">29</a>](C), [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref030" target="_blank">30</a>](D) and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref031" target="_blank">31</a>](E). In (D) and (E) the terminal parts of the ITS2 region with a secondary structure matching the one observed in cryo electron microscopy analyses of 7S-preLSUs shown in (A) and (B) are highlighted in blue. Position of the endonucleolytic C2 processing site (C2) according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref028" target="_blank">28</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179405#pone.0179405.ref049" target="_blank">49</a>] and tethered MNase cleavage sites (black arrows) are indicated.</p

Association of Noc2 with early LSU precursors after <i>in vivo</i> depletion of LSU r-proteins required for early LSU pre-rRNA processing.

<p>The indicated yeast strains, expressing a chromosomally-encoded TAP-tagged version of the LSU biogenesis factor Noc2, together with either the indicated or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter, were cultivated for four hours in glucose-containing medium to shut down expression of the respective LSU r-protein gene. Noc2-TAP and associated pre-ribosomal particles were then affinity purified from the corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (“Input” lanes 1–12) or of parts of the affinity purified fractions (“IP”lanes 13–24) was analyzed by northern blotting. (pre-) rRNAs detected by DNA oligonucleotide probes shown on the right are denoted on the left. Equal signal intensities in the Input and IP fractions indicate that 4% of the respective (pre-)rRNA population co-purified with Noc2-TAP.</p