The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.     

YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification.     

Gateway Role for rRNA Precursors in Ribosome Assembly

In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.     

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.     

The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.     

Era, an essential Escherichia coli small G-protein, binds to the 30S ribosomal subunit.

Era is an essential G-protein in Escherichia coli identified originally as a homologue protein to Ras (E. coli Ras-like protein). It binds to GTP/GDP and contains a low intrinsic GTPase activity. Its function remains elusive, although it has been suggested that Era is associated with the cytoplasmic membrane, cell division, energy metabolism, and cell-cycle check point. Recently, a cold-sensitive phenotype was found to be suppressed by the overexpression of 16S rRNA methyltransferase, suggesting Era association with the ribosome. Here we demonstrate that Era specifically binds to 16S rRNA and the 30S ribosomal subunit. Both GTP and GDP, but not GMP, inhibit Era binding to ribosomal component. Involvement of Era in protein synthesis is suggested by the fact that Era depletion results in the translation defect both in vitro and in vivo.     

The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.     

The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts

The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

The connection between ribophagy, autophagy and ribosomal RNA decay

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.     

The connection between ribophagy,autophagy and ribosomal RNA decay

Ribosomes are essential components of all cells. A large body of knowledge has been accumulated regarding ribosome synthesis and assembly; however, the pathways of normal ribosome turnover, especially rRNA decay, are not known. Some information on ribosome recycling derives from studies on starved yeast cells that use a specialized type of autophagy, called ribophagy, to differentially target ribosomes for degradation. We found that Arabidopsis RNS2, a conserved ribonuclease of the RNase T2 family, is necessary for normal decay of rRNA. Mutants lacking RNS2 activity have longer-lived rRNA, accumulate RNA in the vacuole and show constitutive macroautophagy. Thus, it is clear that normal rRNA decay is necessary to maintain cellular homeostasis. These phenotypes and the subcellular localization of RNS2 in the endoplasmic reticulum and the vacuole suggest that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for recycling under normal growth conditions.     

The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome.     

Identification of the sites of action of SrmB, a DEAD-box RNA helicase involved in Escherichia coli ribosome assembly

DEAD-box RNA-dependent ATPases are ubiquitous enzymes that participate in nearly all processes involving RNA, but their detailed molecular functions remain generally unknown. SrmB, one of the five Escherichia coli DEAD-box proteins, participates in the assembly of the large ribosomal subunit notably by facilitating the incorporation of L13, one of the ribosomal proteins that bind 23S rRNA earliest. Previously, we showed that SrmB is tethered to nascent ribosome through interactions with L4, L24 and the region from domain I of 23S rRNA that binds them. To identify the sites of action of SrmB, we have characterized rRNA mutations that bypass SrmB requirement. Five of them affect the same position from two repeated heptanucleotides in domain II of 23S rRNA, whereas two others affect a complementary hexanucleotide in 5S rRNA. Thus the sites of action of SrmB differ from its tethering site. In the mature ribosome, one of the heptanucleotides participates in a highly compact structure that contacts L13, the '1024 G-ribo wrench'. In addition, we have observed that the assembly defect of ΔsrmB cells worsens as rRNA synthesis increases. Based on these results, we propose two non-exclusive scenarios for the role of SrmB in ribosome assembly.     

Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2'-O-ribose methylation of a conserved rRNA position in yeast

In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites. LSU-Um2918 is one of the four remaining positions. This residue is highly conserved and located in the peptidyl transferase center of the ribosome. The equivalent position on the E. coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase. We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918. When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated. In vitro, Spb1p is able to methylate U2918 on 60S subunits. Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism.     

The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity.

We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).