Mak5 and Ebp2 act together on early Pre-60S particles and their reduced functionality bypasses the requirement for the essential Pre-60S factor Nsa1

Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these ’Mak5 cluster’ factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation

The DEAD-box protein Mak5 is associated with the Nsa1 pre-60S particle.

<p>Mak5 is associated with the early nucleolar Ssf1 and late nucleolar Nsa1 pre-60S particles. The indicated TAP-tagged bait proteins were affinity-purified from cells expressing Mak5-GFP. The final EGTA eluates were analyzed by SDS-PAGE and Coomassie staining (top) and Western blotting using anti-GFP, anti-Nop7, anti-Ebp2 and anti-Rpl3 antibodies (bottom).</p

Over-expression of <i>RIX7</i> enhances the growth defect and 60S subunit deficiency of <i>mak5.R728</i>* and <i>ebp2.K287</i>* mutants.

<p>The MAK5 (<b>A</b>) and EBP2 (<b>B</b>) shuffle strains were co-transformed with plasmids harbouring either the MAK5 or EBP2 wild-type genes or the <i>mak5.R728</i>* or <i>ebp2.K287</i>* mutant alleles and empty vector or plasmids expressing wild-type RIX7 and the dominant-negative <i>RIX7.K580A</i> or <i>RIX7.E634Q</i> alleles under the control of the <i>GAL1-10</i> promoter. After plasmid shuffling on plates containing 5-FoA, cells were restreaked on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp (Glucose) and SGal-Leu-Trp (Galactose) plates, which were incubated at 30°C for 3 d, 3.5 d or 4 d, respectively. (<b>C</b> and <b>D</b>) Over-expression of RIX7 affects 60S subunit biogenesis in <i>mak5.R728</i>* and <i>ebp2.K287</i>* mutant cells. The MAK5 (<b>C</b>) and EBP2 (<b>D</b>) shuffle strains were co-transformed with plasmids harbouring either the MAK5 or EBP2 wild-type genes or the <i>mak5.R728</i>* or <i>ebp2.K287</i>* mutant alleles and empty vector or a plasmid expressing wild-type RIX7 under the control of the <i>GAL1-10</i> promoter. After plasmid shuffling on plates containing 5-FoA, cells were restreaked on SC-Leu-Trp plates. Transformed cells were first pre-grown in SC-Leu-Trp medium, then diluted into SC-Leu-Trp medium with raffinose as carbon source and, finally, expression of wild-type RIX7 was induced by addition of galactose to the medium. After ~16 h of galactose induction, cell extracts were prepared under polysome-conserving conditions and eight A₂₆₀ units were resolved in 10-50% sucrose gradients. The absorption profiles were recorded by continuous monitoring at A₂₅₄. Sedimentation is from left to right. The peaks of free 40S and 60S subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are highlighted by arrowheads. </p

Synthetic lethal interactions between different <i>mak5</i> alleles and <i>ebp2</i>, <i>∆nop16</i>, <i>rpf1</i> and <i>rpl14a</i> alleles.

<p><i>MAK5</i>/<i>EBP2</i> (<b>A</b>), <i>MAK5</i>/<i>∆nop16</i> (<b>B</b>), <i>MAK5</i>/<i>RPF1</i> (<b>C</b>) and <i>MAK5</i>/<i>RPL14A</i> (<b>D</b>) shuffle or double shuffle strains were co-transformed with plasmids harbouring the indicated wild-type and mutant alleles and/or empty vector (YCplac22). Cells were restreaked on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp and SC+5-FoA-Leu-Trp plates, which were incubated for 3 d or 4 d at 30°C (<b>A</b>, <b>B</b>, and <b>C</b>). In the case of the <i>MAK5</i>/<i>RPL14A</i> double shuffle strain, transformed cells were restreaked, after plasmid shuffling on plates containing 5-FoA, on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp plates, which were incubated for 3 d at 30°C (<b>D</b>).</p

Dominant-lethal <i>RIX7</i> alleles retain their negative effect on growth in the absence of Nsa1.

<p><i>MAK5</i>/<i>NSA1</i> (<b>A</b>) and <i>NOP4</i>/<i>NSA1</i> (<b>B</b>) double shuffle strains were co-transformed with plasmids harbouring either the MAK5 or NOP4 wild-type genes or the <i>mak5.R728</i>* or <i>nop4.S460L</i> mutant alleles and plasmids carrying the NSA1 wild-type gene or, expressed under the control of the <i>GAL1-10</i> promoter, wild-type RIX7 and the dominant-negative <i>RIX7.K580A</i> or <i>RIX7.E634Q</i> alleles or empty vector. After plasmid shuffling on plates containing 5-FoA, cells were restreaked on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp (Glucose) and SGal-Leu-Trp (Galactose) plates, which were incubated for 3 d or 4 d at 30°C, respectively.</p

Synthetic lethal interactions between the <i>ebp2.K287</i>* allele and <i>∆nop16</i>, <i>rpf1</i> and <i>rpl14a</i> alleles.

<p><i>EBP2</i>/<i>∆nop16</i> (<b>A</b>), <i>EBP2</i>/<i>RPF1</i> (<b>B</b>) and <i>EBP2</i>/<i>RPL14A</i> (<b>C</b>) shuffle or double shuffle strains were co-transformed with plasmids harbouring the indicated wild-type and mutant alleles and/or empty vector (YCplac22). Cells were restreaked on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp and SC+5-FoA-Leu-Trp plates, which were incubated for 3 d or 4 d at 30°C (<b>A</b> and <b>B</b>). In the case of the <i>EBP2</i>/<i>RPL14A</i> double shuffle strain, transformed cells were restreaked, after plasmid shuffling on plates containing 5-FoA, on SC-Leu-Trp plates and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp plates, which were incubated for 3 d at 30°C (<b>C</b>).</p

The D2 domain of the AAA-ATPase Rix7 harbors the catalytic activity.

<p>(<b>A</b>) Schematic representation of the domain organization of Rix7. N, N-terminal domain (amino acids 1-174); NLS, predicted bipartite nuclear localization signal (amino acids 175-194); D1, AAA-domain D1 (amino acids 208-520); D2, AAA-domain D2 (amino acids 536-823). The amino acid changes within the Walker A and B motifs of D1 or D2, respectively, are indicated. (<b>B</b>) <i>In </i><i>vivo</i> phenotypes of the Walker A and B mutants generated in D1 and D2. Empty vector (YCplac111) and plasmid-borne wild-type RIX7 or the indicated <i>rix7</i> mutant alleles under the control of the authentic promoter were transformed into the RIX7 shuffle strain. Transformants were spotted in 10-fold serial dilution steps onto SC-Leu and SC+5-FoA plates, which were incubated for 5 d at 23°C (upper panel). Expression levels of plasmid-borne TAP-tagged wild-type RIX7 and the indicated <i>rix7</i> Walker A and B mutant alleles, expressed from their cognate promoter in a haploid wild-type strain, were determined in whole cell lysates by Western analysis using anti-ProtA and anti-Arc1 (loading control) antibodies (lower panel). (<b>C</b>) Walker A and B mutations within AAA-domain D2 confer a dominant-lethal phenotype. Empty vector and wild-type RIX7 or the indicated <i>rix7</i> mutant alleles, expressed under the control of the inducible <i>GAL1-10</i> promoter, were transformed into a haploid wild-type strain. Transformants were spotted in 10-fold serial dilution steps onto SC-Leu (Glucose) and SGal-Leu (Galactose) plates, which were incubated for 4 d at 23°C and 30°C, respectively.</p

Model summarizing the genetic networks established in this study.

<p>Nsa1 (green) is a component of late nucleolar pre-60S particles (light bordeaux) whose release and recycling is mediated by the AAA-type ATPase Rix7 (turqoise) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082741#B39" target="_blank">39</a>]. Mutant alleles of the genes encoding the 60S biogenesis factor Ebp2 (red), the DEAD-box RNA helicase Mak5 (light blue), the methyltransferase component of C/D-box snoRNPs Nop1 (chartreuse) and the RRM-containing RNA-binding protein Nop4 (pink) suppress the lethality of <i>∆nsa1</i> null mutant cells. These bypass suppressions are indicated by red double arrows, whose line thickness correlates with the observed strength of the suppression. Mak5 forms a genetic network with Ebp2, Nop16, Rpf1 and the r-protein Rpl14. Synthetic lethal and synthetic enhancement interactions amongst these are depicted by blue continuous or dashed ’negative’ arrows, respectively. We propose that Ebp2, Mak5, Nop16 and Rpf1, referred to as the ’Mak5 cluster’ factors, may orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of the r-proteins Rpl6, Rpl14 and Rpl16 and the rRNA expansion segments ES7L and ES39L (not depicted). Since dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, Rix7 may have additional nuclear substrates besides Nsa1. Finally, over-expression of Rix7 negatively affects growth of <i>mak5</i> and <i>ebp2</i> mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject them to degradation (gray trashcan). We therefore propose that Rix7 may, besides specifically releasing and recycling Nsa1, sense the structural integrity of pre-60S particles and, if these are excessively damaged, channel them into a clearance pathway.</p

Bypass suppression of <i>∆nsa1</i> null lethality by the <i>mak5.R728</i>*, <i>nop1.M232K</i> and <i>nop4.S460L</i> alleles.

<p><i>MAK5</i>/<i>NSA1</i> (<b>A</b>), <i>NOP1</i>/<i>NSA1</i> (<b>B</b>) and <i>NOP4</i>/<i>NSA1</i> (<b>C</b>) double shuffle strains were co-transformed with plasmids harbouring the indicated wild-type and mutant alleles and/or empty vectors (YCplac111 or YCplac22). After plasmid shuffling on plates containing 5-FoA, cells were restreaked on synthetic complete medium lacking leucine and tryptophan (SC-Leu-Trp) and then spotted in 10-fold serial dilution steps onto SC-Leu-Trp plates, which were incubated for 3 d at 30°C, 4 d at 23°C and 4 d at 37°C.</p

Synchronizing nuclear import of ribosomal proteins with ribosome assembly

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes