Yeast UTP B subcomplex reconstitution in insect cells.

<p>All selected UTP B components were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991 or K1992. The protein content of the indicated bands was identified by MS and are indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. (<b>A</b>) Lysates of 2×10⁸ cells infected with K1991were used for two-step affinity purification. Pwp2-TAP was used as the bait protein in the first affinity purification step with IgG-coupled Sepharose resin, and Pwp2-containing components were eluted with TEV protease (Lane 1). Utp6-HA-containing components were purified from 90% of the first elution sample using anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of the eluate (5% each) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. (<b>B</b>) Lysates of 2×10⁸ cells infected with K1992 were used for two-step affinity purification. Utp12-FLAG was purified with anti-FLAG affinity matrix and eluted with the FLAG peptide during the first affinity purification step (Lane 1). A 90% aliquot of the eluted material was used to purify Utp6-HA-containing components with anti-HA affinity matrix, followed by elution with the HA peptide (Lane 2). The composition of both eluates (5%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed by MS. (<b>C</b>) Lysates of 8×10⁷ SF21 cells infected with bacmid K1991 were cleared by the low-speed centrifugation described in the normal protocol (N samples), and half was further cleared by ultracentrifugation (200000×<i>g</i>, 1 h, 4°C, U samples). Pwp2-TAP-containing components were purified from both lysates using IgG-coupled Sepharose resin and eluted with TEV protease. A 10% aliquot of the eluted material was analyzed on a 4-12% gradient SDS-PAGE, stained with Coomassie Blue, and analyzed with MS. (<b>D</b>) Pwp2-TAP-containing components were purified from lysates of 4×10⁷ infected cells (K1991) using IgG-coupled Sepharose resin and TEV elution. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), flow through from the first purification (FT, 0,03%), the eluate from the affinity column (E, 10%), and the fractions from the gel filtration column (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and WB with antibodies against CBP (middle panel) or HA (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification by MS analysis of the corresponding protein is indicated.</p

Yeast tUTP subcomplex reconstitution in insect cells.

<p>All candidate tUTP components were co-expressed in SF21 insect cells infected with baculoviruses containing bacmid K2000. Proteins identified by MS analysis are indicated as Nan1, ▪; Utp10, □; Utp4, ▴; Utp5, ♦; Utp8, •; Utp9, ○ and Utp15, ◊. (<b>A</b>) Two-step affinity purification using two different bait proteins. Lysates of 2×10⁸ infected cells were used in the first affinity purification step to purify Utp15-FLAG-containing component with anti-FLAG affinity matrix which were eluted with the FLAG peptide (Lane 1). 90% of the eluted material was used for the second affinity purification step with anti-HA affinity matrix to purify Nan1-HA containing components, which were eluted with the HA peptide (Lane 2). The composition of both eluates was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. (<b>B</b>) Lysates of 8×10⁷ SF21 cells infected with baculovirus K2000 were cleared by low-speed centrifugation as described (N samples) and half of the sample was further cleared by ultracentrifugation (200000×<i>g</i>, 1 h, 4°C, U samples). Utp15-FLAG-containing components were purified from both lysates using anti-FLAG affinity matrix and eluted with the FLAG peptide. The eluted material (10%) was analyzed on a 4–12% gradient SDS-PAGE, stained with Coomassie Blue, and the protein content of the indicated bands was identified by MS analysis. (<b>C</b>) Utp15-FLAG-containing components were purified from lysates of 4×10⁷ infected cells using anti-FLAG affinity matrix and eluted with the FLAG peptide. Half of the eluate was fractionated on a Superose 6 gel filtration column. Aliquots of the lysate (L, 0,03%), the eluate (E, 10%) and the fractions (2–13; 15%) were analyzed by SDS-PAGE (upper panel) and by WB using antibodies against HA (middle panel) or FLAG (lower panel) epitopes. Elution of marker proteins in independent gel filtration runs are indicated at the top. Correct identification of the corresponding protein by MS analysis is indicated.</p

Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.

<p>Plasmids: Description of plasmids used in this work. Database Number, plasmid backbone used to clone the indicated genes is specified. Original References for previously used plasmids are indicated. When required, plasmids used during the recombination reaction are also indicated.</p

Identification of different UTP B building blocks.

<p>Tagged proteins were purified from cell extracts containing different UTP B components in one or two step affinity purifications. Correct identification by MS analysis of the corresponding protein is indicated as Pwp2, ▪; Utp6, •; Utp12, ♦; Utp13, ◊; Utp18, ○ and Utp21, ▴. Expression of the tagged proteins is indicated as +: untagged protein expressed; T:TAP-tagged; F: FLAG-tagged; *: bait protein. (<b>A</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K1987, K2134, K2135, K2136, K1991 and K1978. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease (Lanes 1–6) or with anti-FLAG affinity beads and elution with FLAG peptide (Lane 7). Samples of the elution were analyzed with SDS-PAGE and MS analysis. (<b>B</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K2137, K2134, K2136, K2138 and K2139. Expression of the tagged proteins is indicated. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with anti-FLAG affinity matrix and eluted with the FLAG peptide. Samples of the elution were analyzed with SDS-PAGE and MS analysis. Note that a band compatible with the size of Utp4-TAP is observed in Lane 3 but was not possible to characterize by MS analysis. (<b>C</b>) Combinations of the indicated proteins were co-expressed in SF21 insect cells infected with baculoviruses containing the bacmids K1991, K2134, K2135, K2136, K2137 and K1987. The bait proteins were purified from lysates of 5×10⁷ infected insect cells with IgG-coupled beads and eluted with TEV protease. Aliquots of the elution (upper panel) or of the corresponding cell lysate (lower panel) were analyzed by WB with anti-HA antibody. The corresponding co-expressed proteins are indicated at the top of the figure.</p

Oligonucleotides: Oligonucleotides used for cloning of Utp genes are described. Database numbers, gene amplified and sequence are indicated.

<p>Oligonucleotides: Oligonucleotides used for cloning of Utp genes are described. Database numbers, gene amplified and sequence are indicated.</p

Refined model of tUTP and UTP B architecture.

<p>Binary interactions observed by protein-fragment complementation assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114898#pone.0114898-Tarassov1" target="_blank">[13]</a> (red line), yeast two hybrid assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114898#pone.0114898-Champion1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114898#pone.0114898-Tarassov1" target="_blank">[13]</a> (blue line) or both (green line) are depicted for tUTP (<b>A</b>) and UTP B (<b>B</b>) components. Arrows point from prey to bait proteins. Building blocks observed in the present study are grouped by solid surfaces for tUTP (<b>A</b>) and UTP B (<b>B</b>) subcomplexes. Loose interaction (yellow surface); whole complex (white surface); Dimer (green surface); Trimer (purple surface); core-complex (red surface); dissociable dimer (blue surface).</p

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

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

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