Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing

The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2′-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2–3 modifications in the A- and P-site regions can cause (1) reduced growth rates (∼15%–50% slower); (2) reduced amino acid incorporation rates (14%–24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation (∼60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.     

Helix 69 in 23S rRNA modulates decoding by wild type and suppressor tRNAs

Helix 69 of 23S rRNA forms one of the major inter-subunit bridges of the 70S ribosome and interacts with A- and P-site tRNAs and translation factors. Despite the proximity of h69 to the decoding center and tRNAs, the contribution of h69 to the tRNA selection process is unclear: previous genetic analyses have shown that h69 mutations increase frameshifting and readthrough of stop codons. However, a complete deletion of h69 does not affect the selection of cognate tRNAs in vitro. To address these discrepancies, the in vivo effects of a range of single- and multi-base h69 mutations in Escherichia coli 23S rRNA on various translation errors have been determined. While a majority of the h69 mutations examined here affected readthrough of stop codons and frameshifting, the ΔA1916 single base deletion mutation uniquely influenced missense decoding. Different h69 mutants had either increased or decreased levels of stop codon readthrough. The h69 mutations that decreased UGA readthrough also decreased UGA reading by a mutant, near-cognate tRNA^Trp carrying a G24A substitution in the D arm, but had far less effect on UGA reading by a suppressor tRNA with a complementary anticodon. These results suggest that h69 interactions with release factors contribute significantly to termination efficiency and that interaction with the D arm of A-site tRNA is important for discrimination between cognate and near-cognate tRNAs.     

rRNA modifications in an intersubunit bridge of the ribosome strongly affect both ribosome biogenesis and activity

The presence of nucleotide modifications in rRNA has been known for nearly 40 years; however, information about their roles is sparse. Here, we describe the consequences of depleting modifications from an intersubunit bridge (helix 69) of the ribosomal large subunit in yeast. Helix 69 interacts with both A and P site tRNAs and contains five modifications. Blocking one to two modifications has no apparent effect on cell growth, whereas loss of three to five modifications impairs growth and causes the broadest defects observed thus far for modification loss in any ribosome region. Major effects include the following: (1) reduced amino acid incorporation rates in vivo (25%-60%); (2) increased stop codon readthrough activity; (3) increased sensitivity to ribosome-based antibiotics; (4) reduced rRNA levels (20%-50%), due mainly to faster turnover; and (5) altered rRNA structure in the ribosome. Taken together, the results indicate that this subset of rRNA modifications can influence both ribosome synthesis and function and in synergistic ways.     

Selection of heptapeptides that bind helix 69 of bacterial 23S ribosomal RNA

Helix 69 of Escherichia coli 23S rRNA has important roles in specific steps of translation, such as subunit association, translocation, and ribosome recycling. An M13 phage library was used to identify peptide ligands with affinity for helix 69. One selected sequence, NQVANHQ, was shown through a bead assay to interact with helix 69. Electrospray ionization mass spectroscopy revealed an apparent dissociation constant for the amidated peptide and helix 69 in the low micromolar range. This value is comparable to that of aminoglycoside antibiotics binding to the A site of 16S rRNA or helix 69. Helix 69 variants (human) and unrelated RNAs (helix 31 or A site of 16S rRNA) showed two- to fourfold lower affinity for NQVANHQ-NH₂. These results suggest that the peptide has desirable features for development as a lead compound for novel antimicrobials.     

RNA aptamers that specifically bind to a 16S ribosomal RNA decoding region construct

RNA–RNA recognition is a critical process in controlling many key biological events, such as translation and ribozyme functions. The recognition process governing RNA–RNA interactions can involve complementary Watson–Crick (WC) base pair binding, or can involve binding through tertiary structural interaction. Hence, it is of interest to determine which of the RNA–RNA binding events might emerge through an in vitro selection process. The A-site of the 16S rRNA decoding region was chosen as the target, both because it possesses several different RNA structural motifs, and because it is the rRNA site where codon/anticodon recognition occurs requiring recognition of both mRNA and tRNA. It is shown here that a single family of RNA molecules can be readily selected from two different sizes of RNA library. The tightest binding aptamer to the A-site 16S rRNA construct, 109.2-3, has its consensus sequences confined to a stem–loop region, which contains three nucleotides complementary to three of the four nucleotides in the stem–loop region of the A-site 16S rRNA. Point mutations on each of the three nucleotides on the stem–loop of the aptamer abolish its binding capacity. These studies suggest that the RNA aptamer 109.2-3 interacts with the simple 27 nt A-site decoding region of 16S rRNA through their respective stem–loops. The most probable mode of interaction is through complementary WC base pairing, commonly referred to as a loop–loop ‘kissing’ motif. High affinity binding to the other structural motifs in the decoding region were not observed.     

Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson–Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5'' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.     

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.     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), and N^6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m⁶A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m⁶A in noncoding RNAs.     

Identification of a novel methyltransferase,Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae

The 25S rRNA of yeast contains several base modifications in the functionally important regions. The enzymes responsible for most of these base modifications remained unknown. Recently, we identified Rrp8 as a methyltransferase involved in m¹A645 modification of 25S rRNA. Here, we discovered a previously uncharacterized gene YBR141C to be responsible for second m¹A2142 modification of helix 65 of 25S rRNA. The gene was identified by reversed phase–HPLC screening of all deletion mutants of putative RNA methyltransferase and was confirmed by gene complementation and phenotypic characterization. Because of the function of its encoded protein, YBR141C was named BMT2 (base methyltransferase of 25S RNA). Helix 65 belongs to domain IV, which accounts for most of the intersubunit surface of the large subunit. The 3D structure prediction of Bmt2 supported it to be an Ado Met methyltransferase belonging to Rossmann fold superfamily. In addition, we demonstrated that the substitution of G180R in the S-adenosyl-l-methionine–binding motif drastically reduces the catalytic function of the protein in vivo. Furthermore, we analysed the significance of m¹A2142 modification in ribosome synthesis and translation. Intriguingly, the loss of m¹A2142 modification confers anisomycin and peroxide sensitivity to the cells. Our results underline the importance of RNA modifications in cellular physiology.     

Pseudouridylation of 23S rRNA helix 69 promotes peptide release by release factor RF2 but not by release factor RF1

Pseudouridine [Ψ] is a frequent base modification in the ribosomal RNA [rRNA] and may be involved in the modulation of the conformational flexibility of rRNA helix-loop structures during protein synthesis. Helix 69 of 23S rRNA contains pseudouridines at the positions 1911, 1915 and 1917 which are formed by the helix 69-specific synthase RluD. The growth defect caused by the lack of RluD can be rescued by mutations in class I release factor RF2, indicating a role for helix 69 pseudouridines in translation termination. We investigated the role of helix 69 pseudouridines in peptide release by release factors RF1 and RF2 in an in vitro system consisting of purified components of the Escherichia coli translation apparatus. Lack of all three pseudouridines in helix 69 compromised the activity of RF2 about 3-fold but did not significantly affect the activity of RF1. Reintroduction of pseudouridines into helix 69 by RluD-treatment restored the activity of RF2 in peptide release. A Ψ-to-C substitution at the 1917 position caused an increase in the dissociation rate of RF1 and RF2 from the postrelease ribosome. Our results indicate that the presence of all three pseudouridines in helix 69 stimulates peptide release by RF2 but has little effect on the activity of RF1. The interactions around the pseudouridine at the 1917 position appear to be most critical for a proper interaction of helix 69 with release factors.     

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (K_ds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (K_d = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.     

Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.     

Evidence for a Competitive-Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA.

We have previously shown that a 5'-terminal region of mouse 5 S rRNA can base-pair in vitro with two distinct regions of 18 S rRNA. Further analysis reveals that these 5 S rRNA-complementary sequences in 18 S rRNA also exhibit complementarity to the Kozak consensus sequence surrounding the mRNA translational start site. To test the possibility that these 2 regions in 18 S rRNA may be involved in mRNA binding and translational initiation, we have tested, using an in vitro translation system, the effects of DNA oligonucleotides complementary to these 18 S rRNA sequences on protein synthesis. Results show that an oligonucleotide complementary to one 18 S rRNA region does inhibit translation at the step of initiation. We propose a Competitive-Displacement Model for the initiation of translation involving the intermolecular base-pairing of 5 S rRNA, 18 S rRNA and mRNA.     

Conserved loop sequence of helix 69 in Escherichia coli 23 S rRNA is involved in A-site tRNA binding and translational fidelity

Ribosomal (r) RNAs play a crucial role in the fundamental structure and function of the ribosome. Helix 69 (H69) (position 1906-1924), a highly conserved stem-loop in domain IV of the 23 S rRNA of bacterial 50 S subunits, is located on the surface for intersubunit association with the 30 S subunit by connecting with helix 44 of 16 S rRNA with the bridge B2a. H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step. To investigate the functional importance of the highly conserved loop sequence (1912-1918) of H69, we employed a genetic method that we named SSER (systematic selection of functional sequences by enforced replacement). This method allowed us to identify and select from the randomized loop sequences of H69 in Escherichia coli 23 S rRNA functional sequences that are absolutely required for ribosomal function. From a library consisting of 16,384 sequence variations, 13 functional variants were obtained. A1912 and U(Psi)1917 were selected as essential residues in all variants. An E. coli strain having 23 S rRNA with a U to A mutation at position 1915 showed a severe growth phenotype and low translational fidelity. The result could be explained by the fact that the A1915-ribosome variant has weak subunit association, weak A-site tRNA binding, and decreased translational activity. This study proposes that H69 plays an important role in the control of translational fidelity by modulating A-site tRNA binding during the decoding process.     

Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit-subunit interactions on the 70S ribosome

The small and large subunits of the ribosome are held together by a series of bridges, involving RNA–RNA, RNA–protein and protein–protein interactions. Some 12 bridges have been described for the Escherichia coli 70S ribosome. In this work, we have targeted for mutagenesis, some of the 16S rRNA residues involved in the formation of intersubunit bridges B3, B5, B6, B7b and B8. In addition to effects on subunit association, the mutant ribosomes also affect the fidelity of translation; bridges B5, B6 and B8 increase decoding errors during elongation, while disruption of bridges B3 and B7b alters the stringency of start codon selection. Moreover, mutations in the bridge B5, B6 and B8 regions of 16S rRNA also correct the growth and decoding defects associated with alterations in ribosomal protein S12. These results link bridges B5, B6 and B8 with the decoding process and are consistent with the recently described location of translation factor EF-Tu on the ribosome and the proposed involvement of h14 in activating Guanosine-5′-triphosphate (GTP) hydrolysis by aminoacyl-tRNA•EF-Tu•GTP. These observations are consistent with a model in which bridges B5, B6 and B8 contribute to the fidelity of translation by modulating GTP hydrolysis by aminoacyl-tRNA•EF-Tu•GTP ternary complexes during the elongation phase of protein synthesis.     

Analysis of the function of <Emphasis Type="Italic">E. coli</Emphasis> 23S rRNA helix-loop 69 by mutagenesis

Background  

The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors.     

Pseudomonas aeruginosa cleaves the decoding center of Caenorhabditis elegans ribosomes

Pathogens such as Pseudomonas aeruginosa advantageously modify animal host physiology, for example, by inhibiting host protein synthesis. Translational inhibition of insects and mammalian hosts by P. aeruginosa utilizes the well-known exotoxin A effector. However, for the infection of Caenorhabditis elegans by P. aeruginosa, the precise pathways and mechanism(s) of translational inhibition are not well understood. We found that upon exposure to P. aeruginosa PA14, C. elegans undergoes a rapid loss of intact ribosomes accompanied by the accumulation of ribosomes cleaved at helix 69 (H69) of the 26S ribosomal RNA (rRNA), a key part of ribosome decoding center. H69 cleavage is elicited by certain virulent P. aeruginosa isolates in a quorum sensing (QS)–dependent manner and independently of exotoxin A–mediated translational repression. H69 cleavage is antagonized by the 3 major host defense pathways defined by the pmk-1, fshr-1, and zip-2 genes. The level of H69 cleavage increases with the bacterial exposure time, and it is predominantly localized in the worm’s intestinal tissue. Genetic and genomic analysis suggests that H69 cleavage leads to the activation of the worm’s zip-2-mediated defense response pathway, consistent with translational inhibition. Taken together, our observations suggest that P. aeruginosa deploys a virulence mechanism to induce ribosome degradation and H69 cleavage of host ribosomes. In this manner, P. aeruginosa would impair host translation and block antibacterial responses.

During infection of the nematode Caenorhabditis elegans by the bacterium Pseudomonas aeruginosa, a bacterial virulence mechanism leads to the cleavage of host ribosomal RNAs at the decoding center, thereby shutting down translation.     

Fragment of mRNA coding part complementary to region 1638–1650 of wheat 18S RNA functions as a translational enhancer

The possible involvement of 18S rRNA fragment 1638–1650, including basements of the helices h44 and h28, as well as nucleotides of the ribosomal decoding site in the cap-independent mode of the initiation of the translation of plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions in the 40S ribosomal subunit. It is found that the sequence that is complementary to the 18S rRNA fragment 1638–1650 is able to enhance the efficiency of the reporter mRNA translation when placed just after the initiation codon. The obtained results indicate that, in the course of the cap-independent mode of the initiation of translation, complementary interactions can occur between the mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site.