Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases

doi:10.1016/S0968-0004(00)01549-8

Trends in Biochemical Sciences

Volume 25, Issue 3, 1 March 2000, Pages 106-110

https://doi.org/10.1016/S0968-0004(00)01549-8 Get rights and content

Abstract

Exonic splicing enhancers (ESEs) are discrete sequences within exons that promote both constitutive and regulated splicing. The precise mechanism by which ESEs facilitate the assembly of splicing complexes has been controversial. However, recent studies have provided insights into this question and have led to a new model for ESE function. Other recent work has suggested that ESEs are comprised of diverse sequences and occur frequently within exons. Ominously, these latter studies predict that many human genetic diseases linked to mutations within exons might be caused by the inactivation of ESEs.

Section snippets

The ‘U2AF-recruitment’ model for ESE function: evidence for and against

Protein–protein interaction screens have indicated that, through their RS domains, the SR-family proteins SC35 and ASF/SF2 can interact with each other, with the SR-related U1 snRNP-70kDa (U1-70K) protein, and with the small (35-kDa) subunit of the heterodimeric U2 snRNP auxiliary factor (U2AF-35/65kDa), which contains a short RS domain8, 9. It had been demonstrated previously that the large subunit of U2AF (U2AF-65kDa), which contains three RRMs and also a short RS domain (Fig. 2), binds to

A role for the SRm160/300 splicing coactivator in ESE function

The recent experiments outlined above indicate that ESE-bound SR proteins can communicate with basal components of the spliceosome through a set of interactions that are separate from those required for the binding of U2AF-65kDa to the polypyrimidine tract. These interactions could be direct, involving contacts with one or more snRNP components, or indirect, through one or more factors that bridge ESE-bound SR proteins and spliceosome components, or both. A candidate for such a bridging factor

Diversity and prevalence of ESEs: implications for human diseases

Besides efforts towards gaining mechanistic insights into how ESEs function, a major goal of recent studies has been to determine the precise nature of sequences that constitute an ESE. Surprisingly, 15–20% of sequences within a randomized 18- or 20-mer, when substituted for an ESE within different exon contexts, promote splicing (22, 23 and references within). Functional SELEX (for ‘systematic evolution of ligands by exponential enrichment’) strategies employing randomized ESE-containing

Acknowledgements

My apologies to many colleagues whose original papers could not be cited owing to space constraints. Alan Cochrane, Adam Eldridge, Debbie Field, Jim Friesen, Lea Harrington, Vicki Lay, Lynne Maquat, Susan McCracken and Juan Valcarcel are thanked for stimulating discussions and comments on the manuscript. Our research is supported by grants from the US Dept of Defense Breast Cancer Research Program, the Canada Foundation for Innovation and the MRC of Canada. B.J.B is the recipient of a MRC of

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Duchenne muscular dystrophy (DMD) is a severe genetic disease caused by the loss of the dystrophin protein. Exon skipping is a promising strategy to treat DMD by restoring truncated dystrophin. Here, we demonstrate that base editors (e.g., targeted AID-mediated mutagenesis [TAM]) are able to efficiently induce exon skipping by disrupting functional redundant exonic splicing enhancers (ESEs). By developing an unbiased and high-throughput screening to interrogate exonic sequences, we successfully identify novel ESEs in DMD exons 51 and 53. TAM-CBE (cytidine base editor) induces near-complete skipping of the respective exons by targeting these ESEs in patients’ induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Combined with strategies to disrupt splice sites, we identify suitable single guide RNAs (sgRNAs) with TAM-CBE to efficiently skip most DMD hotspot exons without substantial double-stranded breaks. Our study thus expands the repertoire of potential targets for CBE-mediated exon skipping in treating DMD and other RNA mis-splicing diseases.
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In eukaryotes, precise pre-mRNA processing, including alternative splicing, is essential to carry out the intricate protein translation process. Both point mutations (that alter the translated protein sequence) and synonymous mutations (that do not alter the translated protein sequence) are capable of affecting the splicing process. Synonymous mutations are known to affect gene expression via altering mRNA stability, mRNA secondary structure, splicing processes, and translational kinetics. In higher eukaryotes, precise splicing is regulated by three weakly conserved cis-elements, 5′ and 3′ splice sites and the branch site. Many other cis-acting elements (exonic/intronic splicing enhancers and silencers) and trans-acting splicing factors (serine and arginine-rich proteins and heterogeneous nuclear ribonucleoproteins) have also been found to enhance or suppress the splicing process. The appearance of synonymous mutations in cis-acting elements can alter the splicing process by changing the binding pattern of splicing factors to exonic splicing enhancers or silencer motifs. This results in exon skipping, intron retention, and various other forms of alternative splicing, eventually leading to the emergence of a wide range of diseases. The focus of this review is to elucidate the role of synonymous mutations and their impact on abnormal splicing mechanisms. Further, this study highlights the function of synonymous mutation in mediating abnormal splicing in cancer and development of X-linked, and autosomal inherited diseases.
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In eukaryotes, maturation of pre-mRNA relies on its precise 3′-end processing. This processing involves co-transcriptional steps regulated by sequence elements and other proteins. Although, it holds tremendous importance, defect in the processing machinery will result in erroneous pre-mRNA maturation leading to defective translation. Remarkably, more than 20 proteins in humans and yeast share homology and execute this processing. The defects in this processing are associated with various diseases in humans. We shed light on the CF IA subunit of yeast Saccharomyces cerevisiae that contains four proteins (Pcf11, Clp1, Rna14 and Rna15) involved in this processing. Structural details of various domains of CF IA and their roles during 3′-end processing, like cleavage and polyadenylation at 3′-UTR of pre-mRNA and other cellular events are explained. Further, the chronological development and important discoveries associated with 3′-end processing are summarized. Moreover, the mammalian homologues of yeast CF IA proteins, along with their key roles are described. This knowledge would be helpful for better comprehension of the mechanism associated with this marvel; thus opening up vast avenues in this area.

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Trends in Biochemical Sciences

Talking PointExonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases

Abstract

Section snippets

The ‘U2AF-recruitment’ model for ESE function: evidence for and against

A role for the SRm160/300 splicing coactivator in ESE function

Diversity and prevalence of ESEs: implications for human diseases

Acknowledgements

Curr. Biol.

Cell

J. Biol. Chem.

Cell

Cell

Am. J. Hum. Genet.

Mutat. Res.

Cell

Trends Biochem. Sci.

J. Biol. Chem.

Splicing of precursors to mRNAs by the spliceosomes

The structure and function of proteins involved in mammalian pre-mRNA splicing

Annu. Rev. Biochem.

The superfamily of arginine/serine-rich splicing factors

RNA

SR proteins and splicing control

Genes Dev.

SR-related proteins and the processing of messenger RNA precursors

Biochem. Cell Biol.

Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation

Annu. Rev. Genet.

Protein–protein interactions and 5′-splice-site recognition in mammalian mRNA precursors

Nature

Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor

Proc. Natl. Acad. Sci. U. S. A.

Talking Point
Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases