A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.
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ABSTRACT:
SUBMITTER: Schutz S
PROVIDER: S-EPMC4161973 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.
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Project description:RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.
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Project description:After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin ?/? pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein.
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Project description:The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5' untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62-K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62-K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62-K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62-K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast. IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62-K70 segment of Rps26 and the 5' untranslated region of mRNA. The data suggested a specific role of the Y62-K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62-K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5' untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62-K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast.
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Project description:Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.
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Project description:During viral infection or cellular stress, cap-dependent translation is shut down. Proteins that are synthesized under these conditions use alternative mechanisms to initiate translation. This study demonstrates that at least two alternative translation initiation routes, internal ribosome entry site (IRES) initiation and ribosome shunting, rely on ribosomal protein S25 (RPS25). This suggests that they share a mechanism for initiation that is not employed by cap-dependent translation, since cap-dependent translation is not affected by the loss of RPS25. Furthermore, we demonstrate that viruses that utilize an IRES or a ribosome shunt, such as hepatitis C virus, poliovirus, or adenovirus, have impaired amplification in cells depleted of RPS25. In contrast, viral amplification of a virus that relies solely on cap-dependent translation, herpes simplex virus, is not hindered. We present a model that explains how RPS25 can be a nexus for multiple alternative translation initiation pathways.
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Project description:Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins.
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Project description:Here we profiled small RNAs from whole cell, cytoplasmic and nuclear extracts from three-week-old Arabidopsis seedlings. We unexpectedly found that nuclear functional hc-siRNAs are predominantly present in the cytoplasm. Samples from Arabidopsis thaliana whole cell, cytoplasmic and nuclear extracts with 3 replicates for each. 9 samples in all.
2012-07-12 | E-GEOD-36500 | biostudies-arrayexpress
Project description:In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis.
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