Molecular Insights into Determinants of Translational Readthrough and Implications for Nonsense Suppression Approaches

Abstract

The fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense and stop codons. However, premature termination codons (PTCs) arising from mutations may, at low frequency, be misrecognized and result in PTC suppression, named ribosome readthrough, with production of full-length proteins through the insertion of a subset of amino acids. Since some drugs have been identified as readthrough inducers, this fidelity drawback has been explored as a therapeutic approach in several models of human diseases caused by nonsense mutations. Here, we focus on the mechanisms driving translation in normal and aberrant conditions, the potential fates of mRNA in the presence of a PTC, as well as on the results obtained in the research of efficient readthrough-inducing compounds. In particular, we describe the molecular determinants shaping the outcome of readthrough, namely the nucleotide and protein context, with the latter being pivotal to produce functional full-length proteins. Through the interpretation of experimental and mechanistic findings, mainly obtained in lysosomal and coagulation disorders, we also propose a scenario of potential readthrough-favorable features to achieve relevant rescue profiles, representing the main issue for the potential translatability of readthrough as a therapeutic strategy.

Keywords: nonsense mutations; premature termination codons; ribosome readthrough.

Figure 1

Initial selection and proofreading activity of the translating ribosome. The insertion of cognate or near-cognate, as well as the rejection of non-cognate, aa-tRNAs is exerted by the ribosome during the initial selection step (left panel, with codon-anticodon interactions depicted in the boxes below). Upon GTP hydrolysis, the cognate aa-tRNA is efficiently accommodated, with formation of peptide bond and progression of protein synthesis with a new aa-tRNA selection step (right panel, upper part). The presence of a near-cognate aa-tRNA, which fails to be accommodated, results in rejection of the aa-tRNA through a proofreading step (right panel, lower part).

Figure 2

Fates of normal or PTC-bearing mRNA transcripts during the first step of translation. The splicing of pre-mRNA into the nucleus results in a mature mRNA transcript bound to exon-junction complexes (EJCs) as well as other key components such as the Upf3 (in the nucleus) and Upf2 (in the cytoplasm) proteins. Once transported into the cytoplasm, the mRNA undergoes a first (pioneer) round of translation. In normal conditions, the ribosome displaces all EJCs, resulting in the replacement of CBC with eIF4E, mRNA circularization and protein synthesis, which proceeds until the natural termination codon (NTC) is reached. If a premature termination codon (PTC) is present, EJCs are not efficiently removed by the ribosome, resulting in recruitment of eRF₁, eRF₃, Upf1 and the SMG1 kinase, which leads to Upf1 phosphorylation and degradation of the UPF1-tagged mRNA.

Figure 3

Translation termination and programmed or drug-induced PTC readthrough. (A) Translation in normal conditions, driven by incorporation of cognate aa-tRNAs, and termination elicited by eRF₁ and eRF₃ at the natural termination codon (NTC). (B) Programmed readthrough, as a result of terminal UGA recoding through incorporation of a selenocysteine-carrying aa-tRNA (tRNA^Sec) mediated by a specialized elongation factor (SelB) and a downstream hairpin structure (selenocysteine inserting sequence, SECIS) recognized by the SECIS-binding protein 2 (SBP2). (C) Readthrough-mediated suppression of a PTC through incorporation of a near-cognate aa-tRNA, which can occur spontaneously or be induced by compounds.

Figure 4

Conformational changes in the decoding center upon tRNA or aminoglycoside binding. Schematic representation of the ribosomal decoding center as derived from PDB 1IBM (A), 1IBL (B), and 1IBK (C) (adapted from [1]). In the native 30S ribosomal subunit, adenines 1492 and 1493 are stacked inside helix 44, and G530 in the syn conformation (A). When the correct codon-anticodon loop, formed upon entering of a cognate aa-tRNA, enters the A site, the A1492 and A1493 residues flip out and G530 switches to the anti-conformation (B). In the presence of paromomycin (PAR), the A1492 and A1493 residues are similarly flipped out into the A site mimicking the presence of a cognate aa-tRNA. Red, G530 loop; orange, helix 44; grey, tRNA anticodon; blue, mRNA codon; green, paromomycin.

Figure 5

Outcome of readthrough as an integrated interplay of molecular determinants. The degree of readthrough of translating ribosome is driven by PTC type, which leads to the differential insertion of a subset of amino acids brought by near-cognate aa-tRNAs. A scenario of the resulting readthrough-mediated protein output is depicted in the right panels (see text for details).