Mitochondrial ribosome assembly in Neurospora crassa. Purification of the mitochondrially synthesized ribosomal protein, S-5.

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].

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Mitochondrial ribosome assembly in Neurospora crassa

Journal of Molecular Biology ◽

10.1016/0022-2836(77)90212-1 ◽

1977 ◽

Vol 116 (2) ◽

pp. 189-205 ◽

Cited By ~ 27

Author(s):

Robert J. LaPolla ◽

Alan M. Lambowitz

Keyword(s):

Neurospora Crassa ◽

Ribosome Assembly ◽

Mitochondrial Ribosome

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Faculty Opinions recommendation of Kinetics and mechanism of mammalian mitochondrial ribosome assembly.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732661020.793542790 ◽

2018 ◽

Author(s):

Alexey Amunts

Keyword(s):

Kinetics And Mechanism ◽

Ribosome Assembly ◽

Mitochondrial Ribosome

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A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)69146-x ◽

1988 ◽

Vol 263 (6) ◽

pp. 2848-2852 ◽

Cited By ~ 2

Author(s):

M T Kuiper ◽

M Holtrop ◽

H Vennema ◽

A M Lambowitz ◽

H de Vries

Keyword(s):

Ribosomal Protein ◽

Neurospora Crassa ◽

Splice Site ◽

Nuclear Gene ◽

Splice Site Mutation ◽

Site Mutation ◽

Gene Encoding ◽

Mitochondrial Ribosomal Protein

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Stepwise maturation of the peptidyl transferase region of human mitoribosomes

10.1101/2021.03.29.437532 ◽

2021 ◽

Author(s):

Tea Lenarcic ◽

Mateusz Jaskolowski ◽

Marc Leibundgut ◽

Alain Scaiola ◽

Tanja Schoenhut ◽

...

Keyword(s):

Quality Control ◽

16S Rrna ◽

Active Site ◽

Critical Role ◽

Ribosomal Subunit ◽

Structural Basis ◽

Ribosome Assembly ◽

Peptidyl Transferase ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. Structural basis of the mammalian mitochondrial ribosome assembly is currently not understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving 7 assembly factors. We discover that NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by MRM2 methyltransferase and quality control interactions with a conserved mitochondrial GTPase MTG2 that contacts the sarcin ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

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Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis

10.1101/2021.10.05.463164 ◽

2021 ◽

Author(s):

Benjamin Pillet ◽

Alfonso Méndez-Godoy ◽

Guillaume Murat ◽

Sébastien Favre ◽

Michael Stumpe ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Translational Regulation ◽

De Novo ◽

Spatial Separation ◽

Ribosome Assembly ◽

Mrna Levels ◽

Ribosomal Assembly ◽

Surplus Production ◽

Regulatory Machinery

AbstractThe biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, prevents the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

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Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5281-5288.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5281-5288

Author(s):

W M Wormington

Keyword(s):

Ribosomal Protein ◽

Mammalian Cells ◽

Ribosomal Proteins ◽

5S Rrna ◽

Yeast Cells ◽

Developmental Expression ◽

Rrna Synthesis ◽

Ribosome Assembly ◽

Subunit Assembly ◽

Ribosomal Protein L5

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

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Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5281 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5281-5288 ◽

Cited By ~ 35

Author(s):

W M Wormington

Keyword(s):

Ribosomal Protein ◽

Mammalian Cells ◽

Ribosomal Proteins ◽

5S Rrna ◽

Yeast Cells ◽

Developmental Expression ◽

Rrna Synthesis ◽

Ribosome Assembly ◽

Subunit Assembly ◽

Ribosomal Protein L5

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.

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