Abstract
Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m6A), 5-methylcytosine (m5C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m5C and m6A 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 m5C, m6A, Ψ 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 m5C 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.
Highlights
Modifications within RNA transcripts are highly abundant and found to be essential for numerous biological processes [1]
The present study aimed to identify the influence of single nucleotide modifications, located within coding sequences of mRNAs, on the translation machinery
Thereby, we directly correlated the presence of nucleotide modifications on peptide synthesis in respect to peptide amounts and amino acid compositions
Summary
Modifications within RNA transcripts are highly abundant and found to be essential for numerous biological processes [1]. More than 140 different modification types in all RNA species have been identified [2]. Thereby, the largest number and diversity of nucleotide modifications have been found in transfer RNAs (tRNAs) [3] which have been reported to be essential for efficient and accurate translation [4,5]. Ribosomal RNAs (rRNAs) mainly harbor pseudouridines ( ) or 2 -O-methylated nucleotides (Figure 1A), which accumulate in functional and structurally conserved regions [6]. Thereby, they are involved in numerous aspects of ribosome assembly and translation. Until now the precise functional roles of these modifications as well as their impact on translation remain unsolved [7,8,9]
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