Olga V. Sergeeva

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m2G966 and m5C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m2G966/m5C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNAfMet to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m2G966 and m5C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.

Modifications of ribosomal RNA: From enzymes to function

Modified nucleosides are present in all kinds of stable RNA molecules, tRNAs being particularly rich in them (Auffinger and Westhof, 1998). Ribosomal RNA (rRNA) from all organisms contains modifications, and there is a correlation between the overall complexity of an organism and the number of modified nucleosides in its rRNA. The rRNA of the most primitive bacteria, such as some Mycoplasma species, may possess only 14 modified nucleosides (de Crecy-Lagard et al., 2007). In Escherichia coli, there are 36 modified nucleosides in rRNA (Table I). Yeast ribosomes possess about one hundred rRNA modifications, human rRNA over two hundred (Ofengand and Fournier, 1998; Decatur and Fournier, 2002). Eukaryotes and archaea use snoRNA guided rRNA modification mechanism. This mechanism allows archaea and eukarya to use a limited number of modification enzymes, mainly pseudouridine synthase and 2′-O-methyltransferase to introduce the majority of their rRNA modifications (Decatur and Fournier, 2002). By contrast, bacteria have developed specific enzymes for each one of the (fewer) modifications they have. Nevertheless, there are many different rRNA modifications in bacteria. Despite intensive study for several decades, many open questions remain regarding the functional role of modified rRNA nucleosides. In this review we will focus on rRNA modifications in E. coli and discuss their possible functions.

What do we know about ribosomal RNA methylation in Escherichia coli

A ribosome is a ribonucleoprotein that performs the synthesis of proteins. Ribosomal RNA of all organisms includes a number of modified nucleotides, such as base or ribose methylated and pseudouridines. Methylated nucleotides are highly conserved in bacteria and some even universally. In this review we discuss available data on a set of modification sites in the most studied bacteria, Escherichia coli. While most rRNA modification enzymes are known for this organism, the function of the modified nucleotides is rarely identified.

How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?

Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions.

mRNA-based therapeutics–Advances and perspectives

In this review we discuss features of mRNA synthesis and modifications used to minimize immune response and prolong efficiency of the translation process in vivo. Considerable attention is given to the use of liposomes and nanoparticles containing lipids and polymers for the mRNA delivery. Finally we briefly discuss mRNAs which are currently in the clinical trials for cancer immunotherapy, vaccination against infectious diseases, and replacement therapy.

N6-Methylated Adenosine in RNA: From Bacteria to Humans.

N6-methyladenosine (m(6)A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m(6)A residues range from RNA-RNA tertiary contacts to RNA-protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m(6)A modification, ways to detect m(6)A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans.

Properties of small rRNA methyltransferase RsmD: Mutational and kinetic study

Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunits involvement in translation.

Ribosome: Lessons of a molecular factory construction

The ribosome is a macromolecular complex responsible for protein biosynthesis. Two subunits of the bacterial ribosome contain three RNA molecules of more than 4000 nt in total and more than 50 proteins. Ribosome assembly is an intricate multistep process, which is vital for the cell. The review summarizes the current concepts of the mechanisms sustaining bacterial ribosome assembly in the cell and in vitro model systems. Some details of assembling this machine are still unknown.

Usage of rRNA-methyltransferase for site-specific fluorescent labeling

The possibility of using an S-adenosylmethionine analog, i.e., pent-2-en-4-ynyl S-adenosylhomocysteine (AduEnYn), as an rRNA methyltransferase cofactor has been investigated. The conditions for the cycloaddition reaction of the fluorescent label to the S-adenosylmethionine analog were chosen. The functional activity of E. coli ribosomes was tested under different conditions. It was found that the introduction of the alkynyl radical occurred successfully and did not affect the functional activity of the ribosome; however, the inactivation of the ribosome occurred during the following cycloaddition reaction.

Possible Role of Escherichia coli Protein YbgI

Proteins containing the NIF3 domain are highly conserved and are found in bacteria, eukaryotes, and archaea. YbgI is an Escherichia coli protein whose gene is conserved among bacteria. The structure of YbgI is known; however, the function of this protein in cells remains obscure. Our studies of E. coli cells with deleted ybgI gene suggest that YbgI is involved in formation of the bacterial cell wall.