ABSTRACT
Mammalian mitochondrial ribosomes contain a set of modified nucleotides, which is distinct from that of the cytosolic ribosomes. Nucleotide m4C840 of the murine mitochondrial 12S rRNA is equivalent to the dimethylated m4Cm1402 residue of Escherichia coli 16S rRNA. Here we demonstrate that mouse METTL15 protein is responsible for the formation of m4C residue of the 12S rRNA. Inactivation of Mettl15 gene in murine cell line perturbs the composition of mitochondrial protein biosynthesis machinery. Identification of METTL15 interaction partners revealed that the likely substrate for this RNA methyltransferase is an assembly intermediate of the mitochondrial small ribosomal subunit containing an assembly factor RBFA.
Subject(s)
Methyltransferases/metabolism , Mitochondria/enzymology , RNA, Ribosomal/metabolism , Ribosome Subunits, Small, Eukaryotic/enzymology , Animals , Cells, Cultured , Methylation , Mice , Mitochondria/metabolism , RNA, Mitochondrial/chemistry , RNA, Mitochondrial/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal, 28S/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/metabolismABSTRACT
Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNAâ tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.
Subject(s)
Adenylate Kinase/metabolism , Frameshifting, Ribosomal , Methyltransferases/metabolism , Nuclear Proteins/metabolism , Nucleoside-Triphosphatase/metabolism , Ribosome Subunits, Small, Eukaryotic/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Adenylate Kinase/chemistry , Adenylate Kinase/genetics , Genotype , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleoside-Triphosphatase/chemistry , Nucleoside-Triphosphatase/genetics , Phenotype , Protein Binding , Protein Conformation , Proteolysis , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Structure-Activity Relationship , Time FactorsABSTRACT
During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.
Subject(s)
Adenosine Triphosphate/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribosome Subunits, Small, Eukaryotic/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Binding Sites , Models, Molecular , Nuclear Proteins/metabolism , RNA Cleavage , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosome Subunits, Small, Eukaryotic/chemistry , Ribosome Subunits, Small, Eukaryotic/metabolismABSTRACT
Maturation of the 40S ribosomal subunit precursors in mammals mobilizes several non-ribosomal proteins, including the atypical protein kinase RioK2. Here, we have investigated the involvement of another member of the RIO kinase family, RioK3, in human ribosome biogenesis. RioK3 is a cytoplasmic protein that does not seem to shuttle between nucleus and cytoplasm via a Crm1-dependent mechanism as does RioK2 and which sediments with cytoplasmic 40S ribosomal particles in a sucrose gradient. When the small ribosomal subunit biogenesis is impaired by depletion of either rpS15, rpS19 or RioK2, a concomitant decrease in the amount of RioK3 is observed. Surprisingly, we observed a dramatic and specific increase in the levels of RioK3 when the biogenesis of the large ribosomal subunit is impaired. A fraction of RioK3 is associated with the non ribosomal pre-40S particle components hLtv1 and hEnp1 as well as with the 18S-E pre-rRNA indicating that it belongs to a bona fide cytoplasmic pre-40S particle. Finally, RioK3 depletion leads to an increase in the levels of the 21S rRNA precursor in the 18S rRNA production pathway. Altogether, our results strongly suggest that RioK3 is a novel cytoplasmic component of pre-40S pre-ribosomal particle(s) in human cells, required for normal processing of the 21S pre-rRNA.
