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  • Review Article
  • Published:

Understanding alternative splicing: towards a cellular code

Nature Reviews Molecular Cell Biology volume 6, pages 386–398 (2005)Cite this article

Key Points

  • Alternative splicing allows individual genes to generate multiple mRNAs. Many of these mRNAs encode functionally distinct protein isoforms, thereby bridging the gap between genome and proteome.

  • Alternative splicing mechanisms have traditionally been investigated using individual model systems, but these approaches are now being complemented by global analyses. The regulation of splicing commonly involves the modulation of early steps in spliceosome assembly. The cis elements comprise splicing enhancers and silencers that can be located in either the exons or the introns and that bind activator and repressor proteins.

  • Sometimes, the presence or absence of a single regulator is sufficient to determine alternative splicing pathways. More commonly, combinations of more widespread factors are involved in the choice of splicing pathway. This gives rise to the concept of 'cellular codes', which are constituted by particular combinations of regulatory factors.

  • Members of the SR protein family can activate splicing by binding to exon splicing enhancers (ESEs). Models for the action of enhancers include the recruitment of the U2 auxiliary factor (U2AF) to weak 3′ splice sites, the interaction with co-activators and direct contact with the RNA at the branch point.

  • Repressors, which are frequently members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, function by making splice sites inaccessible or by promoting the formation of 'dead-end' splicing related complexes. Alternative splicing decisions frequently involve the dynamic antagonism between regulatory activators and repressors.

  • Several global strategies for identifying splicing silencers and enhancers have been successfully deployed. These include RNA-binding SELEX studies, functional SELEX in vitro, cell-based selection assays and computational surveys for the differential enrichment of sequence motifs in expected locations for silencers and enhancers.

  • Methods are being developed to detect complete sets of cellular RNAs with which individual splicing regulators interact. These include assays that detect RNAs to which the factor binds, or alternative splicing events that are affected by depletion of the regulator. A key technical development is the recent introduction of alternative splicing microarrays, which allow parallel analysis of large numbers of alternative splicing events and should facilitate the deciphering of cellular codes.

Abstract

In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms — thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

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Figure 1: Elementary alternative splicing events and regulatory elements.
Figure 2: Mechanisms of enhancement and silencing of alternative splicing.

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Acknowledgements

We thank Juan Valcárcel and Sushma Nagaraja Grellscheid for critically reading the manuscript. Work in the laboratory of C.W.J.S. is supported by a programme grant from the Wellcome Trust. We apologize to those colleagues whose work, for reasons of space constraints, we have been unable to cite directly.

Author information

Authors and Affiliations

  1. Advanced Computational Modelling Centre, and ARC Centre for Bioinformatics, Priestly Building, University of Queensland, 4072, Australia

    Francis Clark

  2. Department of Biochemistry, 80 Tennis Court Road, University of Cambridge, CB2 1GA, UK

    Arianne J. Matlin & Christopher W. J. Smith

Authors
  1. Arianne J. Matlin
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Correspondence to Christopher W. J. Smith.

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Related links

Related links

DATABASES

Entrez Gene

protein phosphatase 2A

Swiss-Prot

hnRNPA1

hnRNPL

KSRP

PTB

SC35

SF2/ASF

SRm160/SRm300

SRp20

SRrp86

U2AF35

U2AF65

FlyBase

Dscam

dsx

fruitless

PSI

RSF1

Sxl

tra

TRA2

U1 70K

FURTHER INFORMATION

ESEfinder

RESCUE-ESE

Alternative Splicing Database (ASD)

Glossary

ENHANCER

A cis-acting RNA sequence in an exon (ESE) or intron (ISE) on which a complex, often containing SR proteins, assembles to promote the use of a weak or regulated splice site.

SILENCER

A cis-acting RNA sequence in an exon (ESS) or intron (ISS) on which a complex, often containing heterogeneous nuclear ribonucleoproteins, assembles to repress the use of a splice site.

'GENOME TILING' MICROARRAY

A type of microarray in which overlapping oligonucleotides are designed to cover a genomic region of interest.

NONSENSE-MEDIATED DECAY

(NMD). A surveillance pathway leading to the targeted degradation of mRNAs that contain premature termination codons. In mammals, termination codons that lie more than 50–55 nucleotides upstream of an exon–exon junction are recognized as premature.

EXON DEFINITION

The initial interaction and stabilization across an exon of factors bound to the 3′ and 5′ splice sites prior to the formation of cross-intron bridging interactions.

SR FAMILY OF PROTEINS

Required for both constitutive and regulated splicing, with a modular structure comprising one or two N-terminal RRMs and a C-terminal arginine/serine-rich (RS) domain.

RNA RECOGNITION MOTIF

(RMM). A protein domain that is frequently involved in sequence-specific single-stranded RNA binding, and consists of a βαββαβ fold with the β strands forming a surface that displays two highly conserved RNP motifs and forms contacts with the RNA.

HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN

(hnRNP). A group of >20 proteins that associate with high-molecular-weight nuclear RNA. Some hnRNPs, such as members of the hnRNPA/B family, shuttle in and out of the nucleus, whereas others are strictly nuclear.

KH

An hnRNPK homology domain that binds to single-stranded RNA, and contains α and β secondary structure elements.

SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE

(snRNP). snRNPs U1, U2, U4, U5 and U6, which contain proteins and U-rich RNA, are core components of the spliceosome.

ARTIFICIAL RECRUITMENT

An experimental approach that allows the analysis of 'effector' domains (repressors or activators), independent of the ability to bind RNA, by fusing an RNA-binding domain (typically, the bacteriophage MS2 coat protein) to the protein or domain of interest, and by introducing cognate RNA-binding sites into the reporter RNA.

BRANCH POINT

An adenosine within a variably conserved 'branch point sequence' upstream of the 3′ splice site that is involved in the first chemical step of splicing. A new 2′–5′ phosphodiester is formed between the branch point and the 5′ end of the intron, concomitant with breakage of the link between the 5′ exon and the intron.

PARALOGUE

A sequence, or gene, that originates from a common ancestral sequence, or gene, by a duplication event.

SELEX

(Systematic evolution of ligands by exponential enrichment). An iterative method for the selection of optimal sites for RNA-binding proteins. Starting from a pool of degenerate cDNAs, sequences are subjected to several rounds of transcription, protein binding and RT-PCR until a consensus sequence emerges.

PRE-mRNP

(or mRNP). A ribonucleoprotein complex, of variable protein composition, that is associated with pre-mRNAs or mRNAs, respectively.

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Matlin, A., Clark, F. & Smith, C. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6, 386–398 (2005). https://doi.org/10.1038/nrm1645

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