Role of internal mRNA modifications in modulating translation elongation

by Sakshi Jain
Doctoral thesis
Date of Examination:2023-10-09
Date of issue:2024-09-26
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Alex Caspar Faesen
Referee:Dr. Sonja Lorenz
Referee:Dr. Katherine E Bohnsack
Persistent Address: http://dx.doi.org/10.53846/goediss-10762

 

 

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Abstract

English

Translation is a highly regulated process that defines the composition of the cellular proteome. Numerous mechanisms are known to fine-tune the translational output at different cellular conditions. Recently, a new regulatory layer of ‘mRNA epitranscriptome’ has been discovered that comprises post-transcriptional modifications of the mRNA that do not change its sequence per se but affects gene expression. The modifications are abundant and found in both coding and untranslated regions of the mRNA at conserved sequences. N6-methyladenosine (m6A) is the most abundant mRNA modification, followed by 5-methylcytidine (m5C), N1-methyladenosine (m1A) and 2’-O-methylribose (2’-O-CH3). These modifications have a dedicated effector machinery that shapes their dynamic presence and regulates mRNA architecture, stability and translatability. However, the precise mechanisms by which mRNA modification attenuate translation remain elusive. In this project, I elucidated how m6A and m5C within internal coding regions of the mRNA modulate translational decoding in a fully reconstituted bacterial translation system using a combination of rapid kinetics, smFRET and single particle cryo-EM techniques. m6A does not impair the initial binding of aminoacyl transfer RNA (aa-tRNA) to the ribosome, however fewer ribosomes complete the decoding process with m6A in the A-site codon. This is due to lower stability of the complexes and enhanced tRNA drop-off. The AAA lysine codon (used to study m6A effects) adopts a π- stacked conformation that is remodeled upon aa-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. The observed effect of m6A is further modulated by amino acid/codon identity, it’s position in the mRNA and by interactions with tRNA anticodon modifications. On the other hand, m5C has a different regulatory effect. m5C does not affect decoding, peptide bond formation and translocation with the cognate aa-tRNA. However, at the 2nd codon position, m5C enhances the probability of near-cognate tRNA decoding due to a m5C·A mismatch in the codon-anticodon complex, thereby inducing amino acid misincorporation. These results highlight how modifications outside the Watson-Crick edge interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs. Detailed insights into the mechanism of m6A-induced translation delay and m5C-induced amino acid misincorporation highlight the significance of different mRNA modifications and their potential as post-transcriptional regulatory marks. Our results provide a benchmark for mechanistic epitranscriptomic studies and can be used for investigating additional unexplored mRNA-regulatory elements.
Keywords: Ribosome; m6A; m5C; mRNA modifications