Pseudouridinylation of mRNA coding sequences alters translation

Abstract

Chemical modifications of RNAs have long been established as key modulators of nonprotein-coding RNA structure and function in cells. There is a growing appreciation that messenger RNA (mRNA) sequences responsible for directing protein synthesis can also be posttranscriptionally modified. The enzymatic incorporation of mRNA modifications has many potential outcomes, including changing mRNA stability, protein recruitment, and translation. We tested how one of the most common modifications present in mRNA coding regions, pseudouridine (Ψ), impacts protein synthesis using a fully reconstituted bacterial translation system and human cells. Our work reveals that replacing a single uridine nucleotide with Ψ in an mRNA codon impedes amino acid addition and EF-Tu GTPase activation. A crystal structure of the Thermus thermophilus 70S ribosome with a tRNAPhe bound to a ΨUU codon in the A site supports these findings. We also find that the presence of Ψ can promote the low-level synthesis of multiple peptide products from a single mRNA sequence in the reconstituted translation system as well as human cells, and increases the rate of near-cognate Val-tRNAVal reacting on a ΨUU codon. The vast majority of Ψ moieties in mRNAs are found in coding regions, and our study suggests that one consequence of the ribosome encountering Ψ can be to modestly alter both translation speed and mRNA decoding.

Regulation of mRNA Stability During Bacterial Stress Responses

“…On the other hand, Ψ modifications on mRNA have been shown to increase mRNA stability in yeast and human cells ( Carlile et al, 2014 ) and in Toxoplasma gondii ( Nakamoto et al, 2017 ). A broad study involving E. coli and human cells found that even a single replacement of U with Ψ in mRNA can interfere with translation ( Eyler et al, 2019 ). Whether these modifications ultimately regulate mRNA stability in bacteria as a response to stress is an open question.…”

Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications

“…Transcriptome-wide profiling of RNA modifications have shifted its focus from high abundant non-coding RNAs to mRNAs of minuscules fraction. RNA modifications exert unpreceded regulation over major aspects of mRNA life such as structure 1,2,3,4 ,stability 5 ,6 decay 7 , translation 8,9,10 microRNA binding 11,12 and altering codon potential 10 . To date, 172 modifications (Modomics) 13 are known to exist in biological systems based on mass spectrometry characterizations.…”

Reverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids

“…1a, b), positional substitution of U with Ψ can increase RNA stability by locally enhancing the macromolecular rigidity 23 . Chemically synthesized Ψ and derivatives thereof are important building blocks for study of C-nucleoside biology as well as for numerous synthetic biology applications based on xenobiotic nucleic acids (XNA) [29][30][31][32][33][34][35][36] . For example, the uniform substitution of U by Ψ can render synthetic mRNA non-immunogenic and was shown to enhance the translation efficiency [30][31][32] .…”