Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

Bussiere, Cyril; Hashem, Yaser; Arora, Sucheta; Frank, Joachim; Johnson, Arlen

doi:10.1083/jcb.201112131

Cited by 70 publications

(112 citation statements)

References 48 publications

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“…It is interesting to note that the final steps of large ribosomal subunit assembly occur around regions important for ribosome function, including the central protuberance that participates in subunit joining, the peptidyl transferase center, and the GTPase activation center. This provides further credence to an emerging theme in ribosomal subunit maturation in which translational capacities of both subunits are evaluated before joining to form 80S ribosomes (Bussiere et al 2012;Strunk et al 2012;Karbstein 2013). Biogenesis of these catalytic RNPs seems to have evolved to save for the finale the most crucial quality control system to inspect their prime reason for existence.…”

Section: Discussionmentioning

confidence: 99%

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

et al. 2014

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Despite having high-resolution structures for eukaryotic large ribosomal subunits, it remained unclear how these ribonucleoprotein complexes are constructed in living cells. Nevertheless, knowing where ribosomal proteins interact with ribosomal RNA (rRNA) provides a strategic platform to investigate the connection between spatial and temporal aspects of 60S subunit biogenesis. We previously found that the function of individual yeast large subunit ribosomal proteins (RPLs) in precursor rRNA (pre-rRNA) processing correlates with their location in the structure of mature 60S subunits. This observation suggested that there is an order by which 60S subunits are formed. To test this model, we used proteomic approaches to assay changes in the levels of ribosomal proteins and assembly factors in preribosomes when RPLs functioning in early, middle, and late steps of pre-60S assembly are depleted. Our results demonstrate that structural domains of eukaryotic 60S ribosomal subunits are formed in a hierarchical fashion. Assembly begins at the convex solvent side, followed by the polypeptide exit tunnel, the intersubunit side, and finally the central protuberance. This model provides an initial paradigm for the sequential assembly of eukaryotic 60S subunits. Our results reveal striking differences and similarities between assembly of bacterial and eukaryotic large ribosomal subunits, providing insights into how these RNA-protein particles evolved.

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Section: Discussionmentioning

confidence: 99%

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

et al. 2014

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“…Briefly, 3 nmol of Lumio TM Green were mixed with 3 nmol of protein at 4°C for 8 h. Subsequently the sample was dialyzed against buffer C containing 50 mM Tris-HCl, pH 7, 250 mM NaCl, 5 mM mercaptoethanol to remove the free dye. The amount of labeled protein was calculated spectrophotometrically using the molar extinction coefficients of 11 FRET Stopped-flow Kinetic Measurements-The experiments were carried out at 28°C in buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM MgCl 2 , 10% glycerol), except for the experiments in the absence of Mg 2ϩ ions that used buffer B (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 10% glycerol). Fluorescence stopped-flow experiments were done using an sx.18MV stopped-flow spectrophotometer (Applied Photophysics, Lestherhead, UK).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, EFL1 has four other domains expected to be very similar to those described for EF-2 except for an unstructured insertion of 160 residues within domain 2 that participates in the interaction with SBDS (14). The function of these domains are unknown but they might undergo large conformational changes similar to those observed for EF-2 during translocation (11). The activity of the GTPases can be modulated by effector biomolecules such as those that accelerate the hydrolysis rate of GTP know as guanine nucleotide activating proteins or by guanine nucleotide exchange factors (GEFs).…”

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confidence: 95%

“…EFL1 probably removes eIF6 from the 60S surface through a conformational change similar to that triggered by EF-2 during elongation in the protein synthesis. Additionally, EFL1 also tests the functionality of the newly synthesized 60S subunits before they enter the pool of translating ribosomes by means of a translocation-like activity (11). As a GTPase, EFL1 has a conserved G-domain in domain 1 composed of the five structural motifs (G1-G5) responsible for the binding of guanine nucleotides.…”

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confidence: 99%

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Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

García-Márquez

Gijsbers

Mora³

et al. 2015

Journal of Biological Chemistry

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Background: EFL1 and the protein mutated in the Shwachman-Diamond syndrome (SBDS) participate in the maturation of the 60S ribosomal subunit. Results: SBDS increases the dissociation constant of EFL1 for GDP 60-fold. SBDS S143L disease mutation disrupts the interaction with EFL1. Conclusion: SBDS favors GDP dissociation from EFL1 and disease mutations abolishe this function. Significance: SBDS disease mutations prevent the nucleotide exchange regulation on EFL1.

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“…Syo1 facilitates the synchronized import of the two 5S rRNA-binding proteins, Rpl5 and Rpl11, to ensure stoichiometric the incorporation of these two proteins into the pre-60S subunits and also works as an assembly platform for the 5S ribonucleoprotein (RNP) (11,12). Release of the anti-association factor Tif6 requires elongation factor-like Efl1 and tRNAlike Sdo1 to confirm the integrity of the P-site (13)(14)(15). Transacting factors on cytoplasmic pre-40S subunits not only prevent them from joining translation (16) but also perform a translation-like cycle to ensure the quality of the 40S subunits prior to actual translation of mRNA (17).…”

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confidence: 99%

The Roles of Puf6 and Loc1 in 60S Biogenesis Are Interdependent, and Both Are Required for Efficient Accommodation of Rpl43

Yang

Ting

Liang

et al. 2016

Journal of Biological Chemistry

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Puf6 and Loc1 have two important functional roles in the cells, asymmetric mRNA distribution and ribosome biogenesis. Puf6 and Loc1 are localized predominantly in the nucleolus. They bind ASH1 mRNA, repress its translation, and facilitate the transport to the daughter cells. Asymmetric mRNA distribution is important for cell differentiation. Besides their roles in mRNA localization, Puf6 and Loc1 have been shown to be involved in 60S biogenesis. In puf6⌬ or loc1⌬ cells, pre-rRNA processing and 60S export are impaired and 60S subunits are underaccumulated. The functional studies of Puf6 and Loc1 have been focused on ASH1 mRNA pathway, but their roles in 60S biogenesis are still not clear. In this study, we found that Puf6 and Loc1 interact directly with each other and both proteins interact with the ribosomal protein Rpl43 (L43e). Notably, the roles of Puf6 and Loc1 in 60S biogenesis are interdependent, and both are required for efficient accommodation of Rpl43. Loc1 is further required to maintain the protein level of Rpl43. Additionally, the recruitment of Rpl43 is required for the release of Puf6 and Loc1. We propose that Puf6 and Loc1 facilitate Rpl43 loading and are sequentially released from 60S after incorporation of Rpl43 into ribosomes in yeast.Almost every process in a cell is mediated by proteins, the products of translating mRNA by ribosomes. Ribosomes are composed of two subunits, a small subunit and a large subunit, 40S and 60S, respectively, in eukaryotic cells. In Saccharomyces cerevisiae, the 40S and 60S subunits are assembled from a large primary 35 S rRNA transcribed by RNA polymerase I. Processing of this rRNA yields 5.8S, 18S, and 25S rRNAs. The 5S rRNA is transcribed by RNA polymerase III separately (see reviews in Refs. 1-4). In yeast, 5S, 5.8S, and 25S (28S in higher eukaryotes) rRNA assemble with 39 ribosomal proteins to make up the 60S subunit, whereas 18S rRNA and 33 ribosomal proteins constitute the 40S subunit (5-7).Ribosome biogenesis is necessary for cell growth and proliferation. The synthesis of ribosome is a complex pathway in which over 200 trans-acting factors are involved. Many of the RNA folding, protein assembly, and maturation events are hierarchical, requiring the correct completion of one event to progress to the next event. This is seen in the assembly of ribosomal proteins onto the RNA in bacterial ribosome assembly (8) as well as in cytoplasmic maturation of the large subunit in yeast (9, 10). In addition, trans-acting factors also perform quality control steps for the assembly of the protein synthesis machinery. Syo1 facilitates the synchronized import of the two 5S rRNA-binding proteins, Rpl5 and Rpl11, to ensure stoichiometric the incorporation of these two proteins into the pre-60S subunits and also works as an assembly platform for the 5S ribonucleoprotein (RNP) (11,12). Release of the anti-association factor Tif6 requires elongation factor-like Efl1 and tRNAlike Sdo1 to confirm the integrity of the P-site (13-15). Transacting factors on cytoplasmic pre-40S subuni...

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Integrity of the P-site is probed during maturation of the 60S ribosomal subunit

Abstract: The P-site of the 60S ribosomal subunit signals to Tif6 via Elf1 during ribosomal maturation, suggesting a quasifunctional check of the integrity of the 60S subunit before the first round of translation.

Cited by 70 publications

References 48 publications

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

A hierarchical model for assembly of eukaryotic 60S ribosomal subunit domains

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein

The Roles of Puf6 and Loc1 in 60S Biogenesis Are Interdependent, and Both Are Required for Efficient Accommodation of Rpl43

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