Protein synthesis in erythroid cells III. Monoribosome and polyribosome function in the cell-free system

doi:10.1016/0005-2787(65)90035-3

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis

Volume 108, Issue 3, 8 November 1965, Pages 434-446

https://doi.org/10.1016/0005-2787(65)90035-3 Get rights and content

Abstract

The capacity of reticulocyte polyribosomes of various size and of 80-S ribosomes to synthesize protein were examined before and after dialysis of ribosomes against buffer solutions free of Mg²⁺. Following dialysis, polyribosomes are partly dissociated with a shift in the maximum endogenous capacity to form protein to polyribosomes of smaller size. Evidence is presented that 80-S ribosomes are heterogeneous with respect to (a) endogenous capacity to synthesize protein, and (b) response to poly-U. 80-S ribosomes were recovered from reticulocytes with little or no endogenous activity and little activity after addition of poly-U. Dialysis yielded 80-S ribosomes with higher levels of endogenous activity and 80-S ribosomes more responsive to poly-U. Dialyzed 80-S ribosomes were competent to synthesize protein with labeled peptides and a molecular weight corresponding to that of globin, without detectable polyribosome formation. In contrast, poly-U formed polyribosomes with 80-S ribosomes.

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Impaired ribosomal subunit association in Shwachman-Diamond syndrome
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Citation Excerpt :
In these systems, complete abrogation of SBDS expression results in increased levels of free 40S and 60S subunits and the appearance of halfmer formation, consistent with a subunit-joining defect. Because these ribosomal features were not observed in SDS patient cells, it was possible that reduced 80S levels might reflect other processes, including decreased ribosomal protein stability29 and/or increased degradation of mature ribosomes.30,31 We therefore modified an in vitro subunit association assay25,26 that takes advantage of the magnesium-dependence of 80S formation from 40S and 60S subunits to directly test for ribosome subunit association defects in human SDS cells.
Shwachman-Diamond syndrome (SDS) is an autosomal-recessive marrow failure syndrome with a predisposition to leukemia. SDS patients harbor biallelic mutations in the SBDS gene, resulting in low levels of SBDS protein. Data from nonhuman models demonstrate that the SBDS protein facilitates the release of eIF6, a factor that prevents ribosome joining. The complete abrogation of Sbds expression in these models results in severe cellular and lethal physiologic abnormalities that differ from the human disease phenotype. Because human SDS cells are characterized by partial rather than complete loss of SBDS expression, we interrogated SDS patient cells for defects in ribosomal assembly. SDS patient cells exhibit altered ribosomal profiles and impaired association of the 40S and 60S subunits. Introduction of a wild-type SBDS cDNA into SDS patient cells corrected the ribosomal association defect, while patient-derived SBDS point mutants only partially improved subunit association. Knockdown of eIF6 expression improved ribosomal subunit association but did not correct the hematopoietic defect of SBDS-deficient cells. In summary, we demonstrate an SBDS-dependent ribosome maturation defect in SDS patient cells. The role of ribosomal subunit joining in marrow failure warrants further investigation.
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4 s + 5 s RNA, 9 s RNA, 18 s RNA, 28 s RNA and polyribosomes, all prepared from rabbit reticulocytes, were injected into living oocytes of the frog Xenopus laevis. Only after injection of 9 s RNA or polyribosomes was haemoglobin-like material formed. Haemoglobin was identified by various criteria: gel filtration, carboxymethyl cellulose chromatography, acrylamide gel electrophoresis and ion exchange chromatography of tryptic peptides. 9 s RNA promoted the formation of globin-like material, whether injected with haemin or without haemin.
It is concluded that when injected into a living cell, the 9 s RNA is fairly stable and has the properties of a haemoglobin messenger. The messenger requires no reticulocyte-specific factors for translation, and the translational machinery of the oocyte will accept the messenger RNA from a totally different cell type, from another species. At all stages of assembly and synthesis haemoglobin molecules are reasonably stable in oocytes.
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^☆: A portion of these data appear in abstract form.

^∗∗: Present address: University of Ibadan, School of Medicine, Ibadan, Nigeria.

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Protein synthesis in erythroid cells III. Monoribosome and polyribosome function in the cell-free system☆

Abstract

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Mol. Biol.

Biochim. Biophys. Acta

J. Mol. Biol.

J. Biol. Chem.

Nature