Ribosome stalk assembly requires the dual-specificity phosphatase Yvh1 for the exchange of Mrt4 with P0.
ABSTRACT: The ribosome stalk is essential for recruitment of translation factors. In yeast, P0 and Rpl12 correspond to bacterial L10 and L11 and form the stalk base of mature ribosomes, whereas Mrt4 is a nuclear paralogue of P0. In this study, we show that the dual-specificity phosphatase Yvh1 is required for the release of Mrt4 from the pre-60S subunits. Deletion of YVH1 leads to the persistence of Mrt4 on pre-60S subunits in the cytoplasm. A mutation in Mrt4 at the protein-RNA interface bypasses the requirement for Yvh1. Pre-60S subunits associated with Yvh1 contain Rpl12 but lack both Mrt4 and P0. These results suggest a linear series of events in which Yvh1 binds to the pre-60S subunit to displace Mrt4. Subsequently, P0 loads onto the subunit to assemble the mature stalk, and Yvh1 is released. The initial assembly of the ribosome with Mrt4 may provide functional compartmentalization of ribosome assembly in addition to the spatial separation afforded by the nuclear envelope.
Project description:Mrt4 is a nucleolar component of the ribosome assembly machinery that shares notable similarity and competes for binding to the 25S rRNA GAR domain with the ribosomal protein P0. Here, we show that loss of function of either P0 or Mrt4 results in a deficit in 60S subunits, which is apparently due to impaired rRNA processing of 27S precursors. Mrt4, which shuttles between the nucleus and the cytoplasm, defines medium pre-60S particles. In contrast, P0 is absent from medium but present in late/cytoplasmic pre-60S complexes. The absence of Mrt4 notably increased the amount of P0 in nuclear Nop7-TAP complexes and causes P0 assembly to medium pre-60S particles. Upon P0 depletion, Mrt4 is relocated to the cytoplasm within aberrant 60S subunits. We conclude that Mrt4 controls the position and timing of P0 assembly. In turn, P0 is required for the release of Mrt4 and exchanges with this factor at the cytoplasm. Our results also suggest other P0 assembly alternatives.
Project description:Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.
Project description:The P0 scaffold protein of the ribosomal stalk is mainly incorporated into pre-ribosomes in the cytoplasm where it replaces the assembly factor Mrt4. In analyzing the role of the P0 carboxyl terminal domain (CTD) during ribosomal stalk assembly, we found that its complete removal yields a protein that is functionally similar to Mrt4, whereas a chimeric Mrt4 containing the P0 CTD behaves more like P0. Deleting the P0 binding sites for the P1 and P2 proteins provoked the nuclear accumulation of P0?AB induced by either leptomycin B-mediated blockage of nuclear export or Mrt4 deletion. This effect was reversed by removing P1/P2 from the cell, whereas nuclear accumulation was restored on reintroduction of these proteins. Together, these results indicate that the CTD determines the function of the P0 in stalk assembly. Moreover, they indicate that in cells lacking Mrt4, P0 and its stalk base partner, the L12 protein, bind to pre-ribosomes in the nucleus, a complex that is then exported to the cytoplasm by a mechanism assisted by the interaction with P1/P2 proteins. Furthermore, in wild-type cells, the presence of nuclear pre-ribosome complexes containing P0 but not L12 is compatible with the existence of an alternative stalk assembly process.
Project description:In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein.
Project description:Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.
Project description:The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1?, P1?, P2? and P2?, which form two heterodimers, P1?/P2? and P1?/P2?, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1?-P2? or P1?-P2? heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1?/P2? in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1?/P2?-P0-P1?/P2? structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.
Project description:Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.
Project description:Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (?P2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (?P1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0?AB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0?AB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0?AB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ?P1, but not P0?AB ribosomes increased upon addition of purified P1?/P2?in vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.
Project description:During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.
Project description:We report here that the acidic ribosomal protein P0 is a component of the membrane-associated Potato virus A (PVA) ribonucleoprotein complex. As a constituent of the ribosomal stalk, P0 functions in translation. Although the ribosomal stalk proteins P0, P1, P2, and P3 are all important for PVA infection, P0 appears to have a distinct role from those of the other stalk proteins in infection. Our results indicate that P0 also regulates viral RNA functions as an extraribosomal protein. We reported previously that PVA RNA can be targeted by VPg to a specific gene expression pathway that protects the viral RNA from degradation and facilitates its translation. Here, we show that P0 is essential for this activity of VPg, similar to eIF4E/eIF(iso)4E. We also demonstrate that VPg, P0, and eIF(iso)4E synergistically enhance viral translation. Interestingly, the positive effects of VPg and P0 on viral translation were negatively correlated with the cell-to-cell spread of infection, suggesting that these processes may compete for viral RNA.