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  • Review Article
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Listening to silence and understanding nonsense: exonic mutations that affect splicing

Key Points

  • A large fraction of point mutations that cause genetic diseases affect splicing efficiency and/or accuracy. Most of these mutations directly affect the canonical consensus sequences that define exon–intron boundaries, and are therefore correctly identified as 'splicing mutations'.

  • Information that is present in the coding regions of genes also contributes to the correct identification of exon–intron boundaries. For example, exonic splicing enhancer (ESE) and silencer (ESS) elements are very prevalent and might be present in most, if not all, constitutive and alternative exons.

  • ESEs are recognized by a class of splicing factors, the SR proteins, that promote splicing by recruiting spliceosomal components to the correct splice sites and/or by antagonizing the action of nearby silencers. Several classes of ESEs, which are recognized by different SR proteins, have been identified, and computational tools are being developed to predict the location, specificity and efficiency of putative ESEs.

  • Because ESEs are embedded in protein-coding sequences, some nonsense, missense and translationally silent point mutations have actually been misclassified. Those mutations that inactivate an ESE can result in partial or complete exon skipping, and can therefore markedly affect the structure or amounts of the expressed protein product.

  • An increasing proportion of point mutations are being reported to affect pre-mRNA splicing. Although reading-frame-dependent mechanisms have been proposed to account for nonsense-associated altered splicing (NAS), in most cases the disruption of ESEs or of RNA secondary structures that are required for proper splicing seems to be the mechanism that leads to exon skipping, regardless of the type of point mutation.

  • Some coding single-nucleotide polymorphisms (cSNPs) can be expected likewise to disrupt elements that are involved in splicing and to exert a subtle effect on splice-site selection, thereby contributing to phenotypic variability, to the variable penetrance of mutations elsewhere in the gene and to cell-type-specific differences in gene expression.

  • The effects of point mutations should be routinely analysed at the mRNA level before drawing conclusions about the importance of the affected amino acid, as their correct classification in terms of the actual mechanism of gene inactivation is essential for understanding structure–function relationships in the corresponding protein, for assessing the phenotypic risk in individuals with familial disease predispositions and for devising new therapies.

Abstract

Point mutations in the coding regions of genes are commonly assumed to exert their effects by altering single amino acids in the encoded proteins. However, there is increasing evidence that many human disease genes harbour exonic mutations that affect pre-mRNA splicing. Nonsense, missense and even translationally silent mutations can inactivate genes by inducing the splicing machinery to skip the mutant exons. Similarly, coding-region single-nucleotide polymorphisms might cause phenotypic variability by influencing splicing accuracy or efficiency. As the splicing mechanisms that depend on exonic signals are elucidated, new therapeutic approaches to treating certain genetic diseases can begin to be explored.

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Figure 1: Classical splicing signals and modes of alternative splicing.
Figure 2: Models of SR protein action in exonic-splicing-enhancer-dependent splicing.
Figure 3: Models of splicing silencing.
Figure 4: Examples of splicing alterations caused by exonic mutations.

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Acknowledgements

We thank M. Hastings for valuable comments on the manuscript and members of our labs for stimulating discussions. Work in the authors' labs is supported by the National Institute of General Medical Sciences, National Institute of Neurological Diseases and Stroke, National Cancer Institute and Andrew's Buddies Corporation (L.C. and A.R.K), and by the Joint Research Board of St Bartholomew's Hospital (S.L.C.).

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Correspondence to Adrian R. Krainer.

Related links

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DATABASES

LocusLink

ATM

BRCA1

DMD

Dscam

eIF4E

eIF4G

FANCC

FBN1

FN1

FTDP17

hnRNP

hnRNP A1

HPRT1

IGHM

MAPT

NF1

PTB

SF2/ASF

SMN1

SMN2

TP53

TRB

U2AF

OMIM

Alzheimer disease

ataxia telangiectasia

Duchenne and Becker muscular dystrophy

Marfan syndrome

neurofibromatosis type 1

spinal muscular atrophy

FURTHER INFORMATION

Adrian Krainer's lab

Encyclopedia of Life Sciences

Spliceosomal machinery

Human Gene Mutation Database

IARC TP53 Mutation Database

Shern Chew's lab

Glossary

PSEUDO-EXON

A pre-mRNA sequence that resembles an exon, both in its size and in the presence of flanking splice-site sequences, but that is never recognized as an exon by the splicing machinery (the spliceosome).

EXON DEFINITION

The recognition of a particular pre-mRNA segment as an exon by the spliceosome. It involves interactions between splice sites on either side of an internal exon or between a splice site and the 5′ cap or the polyadenylation signal of a terminal exon.

SNRNP

(Small nuclear ribonucleoprotein). A particle that is composed of a snRNA and several polypeptides.

SPLICING CO-ACTIVATOR

A protein that mediates splicing enhancement without binding directly to the pre-mRNA.

S100 EXTRACT

A post-nuclear, post-ribosomal cellular fraction that can support in vitro splicing only when complemented with one or more SR proteins.

MINIGENE

A simplified laboratory version of a natural gene that lacks one or more of the gene's exons and introns, or portions of them.

RT-PCR

A type of PCR in which RNA is converted into single-stranded DNA, which is then amplified.

OVERLAP-EXTENSION PCR

A method that involves consecutive PCR reactions with overlapping primers, which is useful to recombine different DNA sequences in vitro.

CONSTITUTIVE EXON

An exon that is always included in the mature mRNA, even in different mRNA isoforms.

CRYPTIC SPLICE SITE

A sequence that matches the 5′ or 3′ splice-site consensus, but is only used as a splice site in the context of a mutation elsewhere in the gene, such as one that inactivates or weakens an authentic splice site.

PURIFYING SELECTIVE PRESSURE

Negative selection that tends to eliminate divergent mutations and maintain the current gene sequence.

CODON-USAGE BIAS

Species-specific frequencies of the alternative codons that specify the same amino acid.

SYNONYMOUS DIVERGENCE

The accumulation of silent mutations due to neutral evolution.

CSNPS

Single-nucleotide polymorphisms that are present in the coding regions of a gene.

PENETRANCE

The proportion of genotypically mutant organisms that show the mutant phenotype.

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Cartegni, L., Chew, S. & Krainer, A. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3, 285–298 (2002). https://doi.org/10.1038/nrg775

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