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Selection on synonymous sites: the unwanted transcript hypothesis

Nature Reviews Genetics (2024)Cite this article

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Abstract

Although translational selection to favour codons that match the most abundant tRNAs is not readily observed in humans, there is nonetheless selection in humans on synonymous mutations. We hypothesize that much of this synonymous site selection can be explained in terms of protection against unwanted RNAs — spurious transcripts, mis-spliced forms or RNAs derived from transposable elements or viruses. We propose not only that selection on synonymous sites functions to reduce the rate of creation of unwanted transcripts (for example, through selection on exonic splice enhancers and cryptic splice sites) but also that high-GC content (but low-CpG content), together with intron presence and position, is both particular to functional native mRNAs and used to recognize transcripts as native. In support of this hypothesis, transcription, nuclear export, liquid phase condensation and RNA degradation have all recently been shown to promote GC-rich transcripts and suppress AU/CpG-rich ones. With such ‘traps’ being set against AU/CpG-rich transcripts, the codon usage of native genes has, in turn, evolved to avoid such suppression. That parallel filters against AU/CpG-rich transcripts also affect the endosomal import of RNAs further supports the unwanted transcript hypothesis of synonymous site selection and explains the similar design rules that have enabled the successful use of transgenes and RNA vaccines.

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Fig. 1: Processes acting on the transcription, processing and filtering of mRNA transcripts, with emphasis on roles of codon content or intronic signals.
Fig. 2: Dinucleotide frequencies of human coding sequences, single-exon coding sequences and genomic DNA, and as expected from mononucleotide frequencies.
Fig. 3: Native human exons predicted to be targeted by the HUSH complex.
Fig. 4: The mode of action of disease-associated synonymous mutations.

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Acknowledgements

The authors thank the Humboldt Foundation for the award of the Humboldt Prize to L.D.H. to enable him to spend time in Germany. S.R. is funded by the Evolution Education Trust.

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Authors and Affiliations

  1. Milner Centre for Evolution, Department of Life Sciences, University of Bath, Bath, UK

    Sofia Radrizzani & Laurence D. Hurst

  2. Milner Therapeutics Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK

    Sofia Radrizzani

  3. MRC Human Genetics Unit, Institute for Genetics and Cancer, The University of Edinburgh, Edinburgh, UK

    Grzegorz Kudla

  4. Max-Delbrück-Center for Molecular Medicine in the Helmholtz Society, Berlin, Germany

    Zsuzsanna Izsvák

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Contributions

L.D.H. and S.R. researched data for the article. All authors contributed substantially to discussion of the content. L.D.H. wrote the article. All authors reviewed and/or edited the manuscript before submission. Z.I. contributed to discussion of issues regarding transposable elements specifically.

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Correspondence to Laurence D. Hurst.

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Supplementary information

Glossary

Codon optimality-mediated RNA decay

A process by which mRNAs with abundant non-optimal codons (those matching rare tRNAs) are subject to decay, probably mediated by slow ribosomal progression.

Codon usage bias

A pattern in which synonymous codons occur at frequencies different from some null expectation (often the null expectation is of equal frequency).

CpG islands

Long (>200 bp) stretches of DNA rich in CpG dinucleotides that commonly occur in the vicinity of mammalian promoters. They are commonly unmethylated if the associated gene is expressed.

Cryptic splice sites

Splice sites within exons away from the canonical splice site that can result in alternative splicing.

Effective population size

(Ne). The number of individuals who an idealized neutrally evolving population would require for it to have properties (such as genetic diversity) equivalent to the real population. Ne is often smaller than the census population size (N). In simple cases, it approximates to the number of breeding individuals.

Exonic splice enhancers

(ESEs). Short (6–8 bp) RNA motifs that enable more accurate splicing, particularly in the vicinity of weak splice sites. ESEs often function as RNA-binding sites for serine–arginine-rich proteins.

Exon-junction complex

A protein complex that forms on a pre-mRNA molecule at the junction between two exons joined by RNA splicing.

Exosome complex

A multi-protein intracellular complex that degrades many types of RNA. In eukaryotes, it is present in the cytoplasm, the nucleus and the nucleolus. In humans, the cytoplasmic form is associated with DIS3L, whereas the nuclear complex contains DIS3. The nuclear form is termed the nuclear exosome complex, that which is targeted by the nuclear exosome targeting (NEXT) complex. The (RNA) exosome complex is not to be confused with exosomes, extracellular vesicles generated by cells.

GC-biased gene conversion

(gBGC). Biased gene conversion describes the recombination of short stretches of genetic material from a donor sequence to an acceptor sequence that is biased as to which is the donor strand and which is the acceptor strand. In gBGC, AT:GC mismatches in recombinant sections are resolved with a preference towards G or C.

G-Quadruplexes

Stable secondary structures formed in guanine-rich DNA and RNA sequences.

Inverted repeat Alu elements

(IRAlus). Single-stranded Alu elements that are followed downstream by their reverse complement. This characteristic often allows inverted repeats to fold on themselves and form double-stranded structures.

LINE1

(L1). LINE elements, of which L1 is an example, are the most abundant class of transposable elements in the human genome, formed by autonomous retrotransposition.

Mutational equilibrium

The frequency (for example, of nucleotides) in a population at which, if only mutation bias and neutral evolution affect the frequency, the frequency does not change.

Neutral evolution

The process by which allele frequency is determined by chance events alone operating on alleles of the same fitness.

No-go mRNA decay

A process by which RNAs with stacks of stalled ribosomes are degraded.

Nonsense-mediated decay

(NMD). A process by which mRNA molecules containing premature stop codons are degraded.

Non-stop RNA decay

A process by which mRNAs without a proper stop codon are identified and targeted for decay. In eukaryotes, this process discharges ribosomes stalled at the 3′ end of mRNAs, directing those mRNAs to the exosome complex.

Processing bodies

Ribonucleoprotein bodies found in the cytoplasm that retain mRNA molecules and contain proteins required for mRNA decay. A similar role is carried out by cytoplasmic stress granules.

Retrogenes

Processed copies of genes formed from reverse transcription of mature mRNA molecules of a parental gene (hence without introns).

Spurious transcripts

Transcripts generated by functionally irrelevant cellular events (such as transcription factor binding to random sequence).

Synonymous mutations

Single base-pair mutations in a protein-coding exon that change the codon to a different but synonymous one and hence do not a priori change the amino acid sequence of the encoded protein.

Translational selection

Selection that favours synonymous mutations owing to their effects on translation, typically assumed to be mediated by faster or more accurate translation. Commonly evidenced by a codon usage bias that favours synonymous codons matching more abundant iso-acceptor tRNAs.

Transposable elements

DNA sequences that can move within genomes and replicate without depending on gene replication of the host cell.

Zinc finger antiviral protein

(ZAP). A protein involved in preventing retroviral infection by binding of CpG-rich sequences and recruitment of the RNA degradation machinery.

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Radrizzani, S., Kudla, G., Izsvák, Z. et al. Selection on synonymous sites: the unwanted transcript hypothesis. Nat Rev Genet (2024). https://doi.org/10.1038/s41576-023-00686-7

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