(A) Affinity purification of Nsa1-TAP from wild-type or mutant cells that were first grown at 23°C and then shifted for 3 h to 30°C

The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-Nop7, anti-Nog1, anti-CBP, anti-Rpl3, anti-Rpp0, anti-Rpl10, and anti-Rps8 antibodies (bottom). The band marked with an asterisk corresponds to Rpp0. The bands corresponding to ribosomal proteins are indicated. (B) rRNA composition of Nsa1-TAP affinity-purified from wild-type or mutant cells that were first grown at 23°C and then shifted for 3 h to 30°C. Aliquots of total RNA and of the final EGTA eluates were resolved on a 1.5% agarose-formaldehyde gel, transferred to a nylon membrane, and analyzed by Northern blotting using the indicated probes to detect pre-rRNA and mature rRNA species. (C) Affinity purification of mature 60S ribosomal subunits via Rpl24A-TAP from wild-type or mutant cells expressing Nsa1-GFP. Cells were first grown at 23°C and then shifted for 3 h to 30°C. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP, anti-CBP, anti-Rpl3, anti-Rpp0, and anti-Rps8 antibodies (bottom). The bands marked with asterisks contain Stm1, Rpl5, Rpp0/Asc1, and Rpl2/Rps1/Rps3/Rps4/Rps0/Rpl8/Rps6, respectively. MW, molecular weight protein standard.<p><b>Copyright information:</b></p><p>Taken from "The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles"</p><p></p><p>The Journal of Cell Biology 2008;181(6):935-944.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426938.</p><p></p

Nop7-TAP, Rix1-TAP, and Kre35-TAP were affinity-purified from wild-type or mutant cells expressing Nsa1-GFP

Cells were first grown at 23°C and then shifted for 3 h to 30°C. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP and anti-Rpl3 antibodies (bottom). The indicated proteins (1–13) were identified by mass spectrometry. White lines indicate that intervening lanes have been spliced out. MW, molecular weight protein standard.<p><b>Copyright information:</b></p><p>Taken from "The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles"</p><p></p><p>The Journal of Cell Biology 2008;181(6):935-944.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426938.</p><p></p

The subcellular localization of the indicated GFP-tagged proteins was assessed by fluorescence microscopy in wild-type and mutant cells that were first grown in yeast peptone dextrose at 23°C and then shifted for 3 h to 30°C

Location of Nsa1-GFP was also determined in and cells that were grown at the permissive temperature of 23°C (top). Bar, 5 μm.<p><b>Copyright information:</b></p><p>Taken from "The AAA ATPase Rix7 powers progression of ribosome biogenesis by stripping Nsa1 from pre-60S particles"</p><p></p><p>The Journal of Cell Biology 2008;181(6):935-944.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426938.</p><p></p

(A) The double-disrupted strains were transformed with the respective plasmid-borne wt or mutant genes

Growth was analyzed by spotting transformants in 10-fold serial dilutions on 5-FOA–containing plates at the indicated temperature for 5 d or on synthetic dextrose complete–Leu-Trp for 3 d (Δ, , and ). No growth indicates synthetic lethality. (B) Schematic representation of the genetic network between and factors involved in transcription-coupled mRNA export. Arrows to gray components indicate synthetic lethality/enhancement, and proteins depicted in white are genetically not linked to .<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A and B) Relative increases (/wt, when wt) or decreases (wt/, when wt) of gene expression were plotted versus their distance to the closest telomere (A) or centromere (B)

A sliding window of 100 genes was used. The average of the 100 genes was used for the y axis, and the distance of the central gene in the window was used for the x axis. The bottom panel of A is an expanded view of the top graph showing the ∼27-kb region close to the telomeres, which exhibits a misregulation in the deletion strain. (C) Expression levels of mRNAs in wt and Δ cells. Total RNA of wt and Δ cells grown in HP and LP was prepared, and cDNA was analyzed by quantitative RT-PCR using specific primers for , , and . Each gene was assayed in triplicates. The mRNA levels of wt HP expression are set as one. One representative dataset of five times independently isolated RNA is shown. Error bars represent SD.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

(A) Schematic overview of pre-mRNA, mRNA, and protein products upon alternative splicing

Either a 126- or a 130-nt intron can be excised by using two alternative 5′ splice sites. In the latter case, a frame shift results in an earlier stop codon and, therefore, in a shorter protein with a different amino acid sequence at the C terminus compared with Src1-L. Conserved domains (HEH/LEM and MSC) and transmembrane domains (M) are indicated. Numbers represent amino acid residues. (B) Whole cell lysates of N- (TAP-Src1) and C-terminal TAP-tagged Src1-L or Src1-S were analyzed by SDS-PAGE followed by Western blotting using anti-ProtA antibodies. (C) Genetic relationship of splice variants with TREX–THO and TREX-2 components. The double-disruption strains were transformed with empty vector, GFP-Src1 splice variants, and the respective TREX component. Transformants were spotted in 10-fold serial dilutions on 5-FOA–containing plates for 5 d at the indicated temperatures.<p><b>Copyright information:</b></p><p>Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"</p><p></p><p>The Journal of Cell Biology 2008;182(5):897-910.</p><p>Published online 8 Sep 2008</p><p>PMCID:PMC2528585.</p><p></p

Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome

<div><p>In eukaryotes, ribosome assembly is a highly complex process that involves more than 200 assembly factors that ensure the folding, modification and processing of the different rRNA species as well as the timely association of ribosomal proteins. One of these factors, Mpp10 associates with Imp3 and Imp4 to form a complex that is essential for the normal production of the 18S rRNA. Here we report the crystal structure of a complex between Imp4 and a short helical element of Mpp10 to a resolution of 1.88 Å. Furthermore, we extend the interaction network of Mpp10 and characterize two novel interactions. Mpp10 is able to bind the ribosome biogenesis factor Utp3/Sas10 through two conserved motifs in its N-terminal region. In addition, Mpp10 interacts with the ribosomal protein S5/uS7 using a short stretch within an acidic loop region. Thus, our findings reveal that Mpp10 provides a platform for the simultaneous interaction with multiple proteins in the 90S pre-ribosome.</p></div

Assembly factor network and reconstitution of the <i>ct</i>Mpp10 module.

<p>(A) One of the top fractions derived from 15–40% sucrose gradient ultracentrifugation containing the free pool of the yeast Mpp10 complex and associated factors, analyzed by SDS-PAGE and Coomassie staining. The sample was first tandem affinity-purified via yeast Imp4-FTpA (Mpp10 factor) and subsequently resolved by sucrose gradient ultracentrifugation. The labeled proteins were identified by mass spectrometry. Degradation products of Imp4 and dimeric version of Mpp10 are marked with a hashtag (#). (B) Systematic Yeast-2-hybrid analysis of core and associated <i>ct</i>Mpp10 assembly factors. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp (permissive), SDC-Leu- Trp-His (selective), and SDC-Leu-Trp-Ade plates (selective for only strong interactions) and grown for 3 days at 30°C. (C) Recombinant co-expression and split tandem-affinity-purification (via pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3) of the <i>ct</i>Mpp10 core complex with stepwise addition of associated factors Utp3 and Rps5/uS7. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). Shown are the corresponding Flag-peptide eluates analyzed by SDS-PAGE and Coomassie staining and Western blotting labeled bands were identified by mass spectrometry. Tags were fused to the N-terminus of the corresponding proteins. (D) Position of the <i>ct</i>Mpp10 factors <i>ct</i>Imp3 and <i>ct</i>Imp4 within the 90S cryo-EM density revealing their relative position to the U3 snoRNA (orange) and pre-18S rRNA domains (green, cyan, and purple). Density map of the <i>C</i>. <i>thermophilum</i> 90S pre-ribosome (EMDB: EMD-8143) and respective fitted <i>ct</i>Mpp10 complex members (PDB: 5JPQ) are visualized using Chimera. A magnification of the fitted models for <i>ct</i>Imp3 (blue), <i>ct</i>Imp4 (turquois), and <i>ct</i>Rps5/uS7 (yellow) is shown in a circle, illustrating their close neighborhood within the particle.</p

Identification of the minimal Rps5-binding motif within Mpp10.

<p>(A) Yeast 2-hybrid analysis of full-length <i>ct</i>Rps5 tested with four indicated fragments of <i>ct</i>Mpp10. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp, SDC-Leu-Trp-His, and SDC-Leu-Trp-Ade plates and grown for 3 days at 30°C. (B) Size exclusion chromatography (SEC) of the reconstituted GST-<i>ct</i>Mpp10(283–332)-<i>ct</i>Rps5 heterodimer. Based on the elution profile recorded at 280 nm, we analyzed fractions 12–19 by SDS-PAGE and Coomassie staining. The complete elution profile (in ml) using a Superdex 200 10/300 column is shown underneath, with indication of the region corresponding to fractions 12–19. The complex was recombinantly co-expressed in <i>E</i>. <i>coli</i>, purified via GST pull-down and eluted with glutathione (GSH). Labeled bands were verified by mass spectrometry. (C) Assembly of the Mpp10 module by recombinant overexpression of the <i>C</i>. <i>thermophilum</i> counterparts, in <i>S</i>. <i>cerevisiae</i>, followed by split-tag affinity-purification using pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3, as first and second bait respectively. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). FLAG eluates were subject to SDS-PAGE followed by Coomassie staining and Western blot analysis using anti-Rps5 and anti-FLAG antibodies.</p

Two hydrophobic patches within the N-terminal region mediate the direct interaction between Mpp10 and Utp3.

<p>(A) Yeast 2-hybrid analysis of full-length <i>ct</i>Utp3 tested with four indicated fragments of <i>ct</i>Mpp10. The indicated constructs were N-terminally fused to either GAL4 activation domain (AD) or GAL4 binding domain (BD). Yeast transformants were spotted onto SDC-Leu-Trp, SDC-Leu-Trp-His, and SDC-Leu-Trp-Ade plates and grown for 3 days at 30°C. (B) Recombinant GST and GST-<i>ct</i>Mpp10 truncations were co-expressed with <i>ct</i>Utp3 in <i>E</i>. <i>coli</i>, and subsequently bound to glutathione resin. GSH-eluates were analyzed by SDS-PAGE followed by Coomassie staining. Labeled bands were identified by mass spectrometry. Lower bands correspond to degradation products and are marked with a hashtag (#). (C) Recombinant overexpression of the <i>C</i>. <i>thermophilum</i> Mpp10 complex components in <i>S</i>. <i>cerevisiae</i>, followed by split-tag affinity-purification using pA-TEV-<i>ct</i>Mpp10 and Flag-<i>ct</i>Imp3, as first and second bait, respectively. The complexes were assembled by over-expression of indicated protein combinations in yeast (pGAL, high-copy plasmids). In this case, we additionally used an N-terminally HA tagged version of <i>ct</i>Utp3. FLAG eluates were subject to SDS-PAGE followed by Coomassie staining and Western blot analysis using anti-HA and anti-FLAG antibodies.</p