Mitochondrial Small Ribosomal Subunit Research Articles

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

Mitochondrial and nuclear mutations that affect the biogenesis of the mitochondrial ribosomes of yeast

1. Several nuclear mutants have been isolated which showed thermo- or cryo-sensitive growth on non-fermentable media. Although the original strain carried mitochondrial drug resistance mutations (CR, ER, OR and PR), the resistance to one or several drugs was suppressed in these mutants. Two of them showed a much reduced amount of the mitochondrial small ribosomal subunit (37S) and of the corresponding 16S ribosomal RNA. Two dimensional electrophoretic analysis did not reveal any change in the position of any of the mitochondrial ribosomal proteins. However one of the mitochondrial ribosomal proteins. However one of the mutants showed a striking decrease in the amounts of three ribosomal proteins S3, S4 and S15. 2. Four temperature-sensitive mitochondrial mutations have been localized in the region of the gene coding for the large mitochondrial ribosomal RNA (23S). These mutants all showed a marked anomaly in the mitochondrial large ribosomal subunit (50S) and/or the corresponding 23S ribosomal RNA.

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.

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].

Nuclear suppressors of the [poky] cytoplasmic mutant in Neurospora crassa. III. Effects on other cytoplasmic mutants and on mitochondrial ribosome assembly in [poky

We have previously isolated six non-allelic, nuclear mutations (sui loci) that partially suppress the growth, respiratory and cytochrome abnormalities of the extranuclear [poky] mutant. A comparison of the mitochondrial ribosome profiles of suppressed and unsuppressed [poky] strains revealed that five of the six suppressors alleviate at least partially the deficiency of mitochondrial small ribosomal subunits that is associated with the [poky] genotype. Six independently isolated Group 1 extranuclear mutants, namely [exn-1], [exn-2], [exn-4-a1, [stp-b1], [SG-1] and [SG-3-A1, which have growth and cytochrome phenotypes similar to [poky] also were found to be deficient in small subunits of mitochondrial ribosomes. Using cytochrome aa3 and b production as a criterion for mitochondrial protein synthesis, it could be shown that the nuclear su I suppressors of [poky] also suppress the other six Group I extranuclear mutants. However, differences in the efficiencies of suppression by suI suppressors suggest that at least some of Group I extrachromosomal mutants are not simply re-isolates of [poky], but represent distinct extranuclear mutations.

The placenta--an environmental problem.

<h3>Summary</h3> Mitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA, whilst acquiring unique protein subunits located on the periphery of the ribosomal subunit structures. We set out to investigate the functional properties of all 14 unique (mitochondrial-specific or supernumerary) human mitoribosomal proteins in the small subunit. Using genome editing with CRISPR-Cas9, we made knockouts for each subunit in HEK293 cells to study the effect on mitoribosome assembly and function in protein synthesis. Unexpectedly, we show that each supernumerary knockout leads to a unique mitoribosome assembly defect with variable impact on mitochondrial protein synthesis. Our data demonstrates that all supernumerary subunits are essential structural components except mS37. Surprisingly, we found the stability of mS37 was reduced in all our supernumerary knockouts of the small and large ribosomal subunits as well as patient-derived lines with mitoribosome assembly defects. We identified that a redox regulated CX₉C motif in mS37 was essential for protein stability, suggesting a potential mechanism to regulate mitochondrial protein synthesis. Together, our findings support a modular assembly of the human mitochondrial small ribosomal subunit mediated by essential supernumerary subunits and identify a redox regulatory role involving mS37 in mitochondrial protein synthesis in health and disease.