Dataset Information

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

ABSTRACT: Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (?) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m(5)C, m(6)A, ? or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs.

SUBMITTER: Hoernes TP

PROVIDER: S-EPMC4737146 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

Hoernes Thomas Philipp TP Clementi Nina N Faserl Klaus K Glasner Heidelinde H Breuker Kathrin K Lindner Herbert H Hüttenhofer Alexander A Erlacher Matthias David MD

Nucleic acids research 20151117 2

Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced ...[more]

PMID: 26578598

Similar Datasets

Project description:BackgroundTrans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes.ResultsComplexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity.ConclusionOur observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.

Project description:Prokaryotic genomes encode a plethora of small noncoding RNAs (ncRNAs) that fine-tune the expression of specific genes. The vast majority of known bacterial ncRNAs are encoded from within intergenic regions, where their expression is controlled by promoter and terminator elements, similarly to protein-coding genes. In addition, recent studies have shown that functional ncRNAs can also be derived from gene 3' untranslated regions (3'UTRs) via an alternative biogenesis pathway, in which the ncRNA segment is separated from the mRNA via RNase cleavage. Here, we report the detection of a large set of decay-generated noncoding RNAs (decRNAs), many of which are completely embedded within protein-coding mRNA regions rather than in the UTRs. We show that these decRNAs are "carved out" of the mRNA through the action of RNase E and that they are predicted to fold into highly stable RNA structures, similar to those of known ncRNAs. A subset of these decRNAs is predicted to interact with Hfq or ProQ or both, which act as ncRNA chaperones, and some decRNAs display evolutionarily conserved sequences and conserved expression patterns between different species. These results suggest that mRNA protein-coding regions may harbor a large set of potentially functional small RNAs.IMPORTANCE Bacteria and archaea utilize regulatory small noncoding RNAs (ncRNAs) to control the expression of specific genetic programs. These ncRNAs are almost exclusively encoded within intergenic regions and are independently transcribed. Here, we report on a large set ncRNAs that are "carved out" from within the protein-coding regions of Escherichia coli mRNAs by cellular RNases. These protected mRNA fragments fold into energetically stable RNA structures, reminiscent of those of intergenic regulatory ncRNAs. In addition, a subset of these ncRNAs coprecipitate with the major ncRNA chaperones Hfq and ProQ and display evolutionarily conserved sequences and conserved expression patterns between different bacterial species. Our data suggest that protein-coding genes can potentially act as a reservoir of regulatory ncRNAs.

Dataset Information

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

Publications

Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets