Review
Alternative splicing: combinatorial output from the genome

https://doi.org/10.1016/S1367-5931(02)00320-4Get rights and content

Abstract

Alternative splicing has emerged as a mechanism that can account for a large proportion of the disparity between the modest number of genes in the human genome and the much higher complexity of the expressed proteome. At least a third, and probably the majority, of human genes are alternatively spliced, and some genes can generate thousands of protein isoforms by complex alternative splicing events. Analysis of the transcriptome will therefore require the development of massively parallel technologies that are able to encompass the complexity arising from alternative splicing.

Introduction

Proteins can provide a structural framework for extreme molecular diversity, as illustrated by antibodies and phage-display technology. A number of recent findings have pointed to alternative pre-mRNA splicing as a generator of protein diversity that in some cases rivals the immune system in the degree of diversity generated by a single gene, and which is widespread throughout multicellular organisms. The relatively limited number of genes revealed by the human genome sequencing projects 1•., 2., 3. compared with the 10–100 fold greater complexity of the expressed proteome (the complete set of proteins in a cell or organism) [4], have underscored the importance of alternative splicing in gene expression. This review focuses upon alternative splicing as a mechanism for combinatorial gene output. Combinatorial input to the control of alternative splicing is covered in other recent reviews 5., 6., 7..

Section snippets

Splicing

Pre-mRNA splicing is an essential step in the expression of most eukaryotic genes, which contain non-coding intron sequences. The introns are copied into the initial pre-mRNA, but are then removed and the exons are ligated together to form translatable mRNA. Pre-mRNA splicing involves the initial recognition of conserved splice-site sequences at the exon–intron boundaries by various protein-splicing and RNA-splicing factors. This leads to the step-wise assembly of the spliceosome, the 60S

Alternative splicing; one gene, more than one protein

Real genes are more complex than the model single-intron substrates used to investigate the mechanisms of splicing. They typically contain multiple introns, and in many cases the exons can be joined in more than one way to generate multiple mRNAs, encoding distinct protein isoforms (Fig. 1). This process — alternative splicing — allows the optional inclusion or substitution of some exons within the constant framework provided by constitutive exons. The resultant protein isoforms commonly vary

The rule, not the exception

In his 1993 Nobel lecture, Phil Sharp speculated that up to 5% of genes were alternatively spliced [9]. Subsequent expressed-sequence tag (EST) and genome-sequencing projects have enabled better-informed estimates, which are uniformly higher. Recent studies find evidence for alternative splicing in at least a third of human genes 1•., 21., 22., 23., 24•., 25., 26••.. The most comprehensive analysis [26••] aligned full-length mRNAs against the whole genome, and used stringent criteria to

Combinatorial output

Simple cases of alternative splicing involve production of two isoforms by use of optional cassette exons or introns, by pairs of mutually exclusive exons or by competing 5′ or 3′ splice sites (Fig. 1). Combinations of these events within individual genes allow the generation of multiple isoforms (Fig. 2). Although individual alternative splicing events within a gene are often co-regulated, in some cases, different combinations of events do not seem to be restricted. ‘Combinatorial splicing’

Observing and understanding the mRNA diversity generated by alternative splicing

The advent of genomic biology, with its shift of emphasis to global, hypothesis-free observation, leads to a new reason for analyzing alternative splicing — comprehension of the entire complement of mRNAs for any given cell or organism, or ‘transcriptomics’. The challenge for genome biology is to understand the functions of all the genes of an organism and how they work together to produce life (Fig. 3). A fundamental requirement of this task is the definition of the repertoire of possible

Conclusions

Alternative pre-mRNA splicing is emerging as a widespread mechanism for modulating the function of the genome. It appears increasingly likely that the majority of human genes produce multiple different proteins — and have multiple activities — through this process. To understand the function of the genome it is therefore essential to identify the full range of mRNAs it can produce, and to develop tools for discovering their function and monitoring their expression.

Update

Modrek and Lee [58••] have written an excellent review on genome-wide bioinformatic analyses of alternative splicing, including a detailed discussion of the limitations of these analyses and references to the resulting online databases. Two recent databases not appearing in the this review are the Putative Alternative Splicing database (PALSdb) [59] and SpliceNest [60]. For PALSdb, the longest mRNA sequences from 19 936 human and 16 615 mouse Unigene clusters were aligned with EST sequences.

Acknowledgements

We thank Juan Valcárcel and Katia Smith-Litière for critically reading the manuscript and Brenton Graveley and Javier Cáceres for providing preprints. Work on alternative splicing in the CWJS laboratory is funded by a grant from the Wellcome Trust (059879).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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