Abstract
RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs. mRNA modifications within coding sequences can either directly or indirectly interfere with protein synthesis. In order to investigate the roles of various natural occurring modified nucleotides, we site-specifically introduced them into the coding sequence of reporter mRNAs and subsequently translated them in HEK293T cells. The analysis of the respective protein products revealed a strong position-dependent impact of RNA modifications on translation efficiency and accuracy. Whereas a single 5-methylcytosine (m5C) or pseudouridine (Ψ) did not reduce product yields, N1-methyladenosine (m1A) generally impeded the translation of the respective modified mRNA. An inhibitory effect of 2′O-methlyated nucleotides (Nm) and N6-methyladenosine (m6A) was strongly dependent on their position within the codon. Finally, we could not attribute any miscoding potential to the set of mRNA modifications tested in HEK293T cells.
Highlights
The idea that mRNAs are simple, static carriers of a plain four-letter code was challenged by the discovery of an increasing number of modified nucleotides within mRNAs
The present study aimed to investigate the potential of some described mRNA modifications study aimed to investigate the potential of somean described mRNA strategy modifications (Figure 1A)The to present intervene with translation elongation
The modified mRNAs were predominantly tested in various eukaryotic in vitro translation systems, in this study HEK293T cells predominantly tested in various eukaryotic in vitro translation systems, in this study HEK293T cells were employed
Summary
The idea that mRNAs are simple, static carriers of a plain four-letter code was challenged by the discovery of an increasing number of modified nucleotides within mRNAs (reviewed in [1,2]). These modifications, found in the untranslated regions (UTRs) as well as in the coding sequences (CDSs), have the capability to add an additional layer of information to the genetic code, thereby expanding the organisms’ toolkit of regulating gene expression. Due to novel refinements in mass spectrometry (MS) and high throughput sequencing, their number and their respective positions seem to change constantly. In addition to the current discussion about the number and the positions of the identified modifications, their significance and the role of numerous modifications are currently debated [24,25]
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