How to avoid undesirable interactions during ribosome assembly?

Ribosome subunit assembly in bacteria is assisted by several non‐ribosomal proteins, the absence of which leads to assembly defects. The two DEAD‐box RNA helicases SrmB and DeaD/CsdA are required for efficient assembly of the ribosome large subunit, in particular at low temperature, but their sites of action on rRNA were not known until now. In this issue of Molecular Microbiology, Proux et al. show that SrmB acts far away from its tethering site on the assembly intermediate particle. A genetic screen identified mutations in complementary sequences of 23S and 5S rRNA that help to bypass SrmB deficiency, partially correcting the large subunit assembly defect. The results suggest that 5S rRNA and 23S rRNA can interact via base‐pairing, forming a non‐native structure that needs to be corrected. The authors discuss attractive hypotheses on SrmB acts during large subunit assembly.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

Functional interdependence among ribosomal elements as revealed by genetic analysis

Summary Escherichia coli strains with preexisting ribosomal mutations were used in order to isolate further ribosomal mutations. The ribosomal mutations used were resistance to erythromycin, spectinomycin, streptomycin or kasugamycin. These mutations cause alteration of specific ribosomal elements, L4, S5, S12 proteins and 16S rRNA respectively. Mutations have been introduced into strains carrying one, two or three of these mutations. Strains with all possible combinations of these four mutations were constructed. The phenotypes of all isolated mutants were tested, and frequently the strains lost one or more of their pre-existing resistances.Thus, functional interactions were revealed among proteins, as well as RNA and proteins within the 30 S ribosomal subunit and as well as between the 30 S and the 50 S ribosomal subunits.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure.

Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.