Expansion of the eukaryotic proteome by alternative splicing

Abstract

The collection of components required to carry out the intricate processes involved in generating and maintaining a living, breathing and, sometimes, thinking organism is staggeringly complex. Where do all of the parts come from? Early estimates stated that about 100,000 genes would be required to make up a mammal; however, the actual number is less than one-quarter of that, barely four times the number of genes in budding yeast. It is now clear that the 'missing' information is in large part provided by alternative splicing, the process by which multiple different functional messenger RNAs, and therefore proteins, can be synthesized from a single gene.

Figure 1. Types of alternative splicing

There are four basic types of alternative splicing: alternative 5′ splice-site selection (a), alternative 3′ splice-site selection (b), cassette-exon inclusion or skipping (c) and intron retention (d). The rectangles in the centre represent pre-mRNAs. For each pre-mRNA, the black lines span the regions that can be spliced out, with the lines above corresponding to the mature mRNA shown on the left and the lines below to the mRNA on the right. That is, the mRNA that is synthesized when the central exon (or intron in d) is skipped is shown on the left, and the mRNA that is synthesized when this sequence is included is shown on the right. In d, the pink portion is considered an exon when included (right) and an intron when skipped (left).

Figure 2. The generation of diverse mRNA repertoires

a, Human KCNMA1 pre-mRNA is depicted, with constitutive exons in blue and alternative exons in yellow and red. The possible splicing patterns for each exon are indicated above and below the pre-mRNA (black lines).The KCNMA1 pre-mRNA contains multiple alternative 5′ splice sites, alternative 3′ splice sites and sequence corresponding to cassette exons. Together, these allow more than 500 mRNA isoforms to be synthesized from a single pre-mRNA. b, Drosophila melanogaster Dscam pre-mRNA is depicted, with constitutive exons in blue and alternative exons in the exon 4, 6, 9 and 17 clusters shown in yellow, orange, red and purple, respectively. The splicing pattern for one mRNA isoform is shown as an example. The Dscam gene contains 95 alternative exons organized into four clusters of mutually exclusive exons (that is, only exon from each cluster is transcribed). These four clusters are at exon 4, exon 6, exon 9 and exon 17, which contain 12, 48, 33 and 2 variable exons, respectively. In combination with the 20 constitutive exons of Dscam, this structure allows 38,016 mRNAs to be synthesized from a single pre- mRNA. c, The organization of the D. melanogaster gene mod(mdg4) is depicted, with common exons in blue and variable exons in purple. The common exons and some of the variable exons are present on one strand of the DNA and are transcribed from left to right as shown, whereas another set of the variable exons is present on the opposite strand and is transcribed from right to left as illustrated. The precise number, and the precise positions of the beginnings and ends, of the common exon pre-mRNA and the variable exon pre-mRNAs is unknown. The possible splicing patterns for each exon in the corresponding pre-mRNA are indicated by black lines, with the possible positions of poly(A)+ tails indicated by green wavy lines. A total of 28 mod(mdg4) mRNAs are synthesized, and this occurs through the trans-splicing of the common exons to one set of variable exons.

Figure 3. Alternative splicing regulatory mechanisms

a, Kinetic effect of splicing silencers on alternative splicing. Left, when two competing 5′ splice sites are present in a hypothetical pre-mRNA and the appropriate silencer is absent (that is, the hnRNP, which silences transcription by binding to the exonic splicing silencer (ESS)), the U1 small nuclear RNP (U1 snRNP) binds to both 5′ splice sites (while U2 snRNP and U2AF bind to the single 3′ splice site), and the proximal 5′ splice site is preferentially used (solid black line; dashed black line indicates splicing to the distal 5′ splice site). Top right, by comparison, when the hnRNP is present, the rate at which the proximal 5′ splice site can be used decreases, promoting splicing to the distal 5′ splice site. Centre right, in comparison with the case on the left, in the absence of a competing 5′ splice site upstream, the presence of the hnRNP does not significantly decrease the rate of splicing. Bottom right, in comparison with the case on the left, when silencer sequences are present near both 5′ splice sites, the hnRNP has no detectable impact on the ratio at which either splice site is used. b, Interplay between transcription elongation rate, chromatin structure and histone modifications, and their impact on alternative splicing. A hypothetical gene is depicted; three exons are shown packaged into nucleosomes. The constitutively transcribed exons (green) are packaged into nucleosomes that constitutively contain histone H3 that is trimethylated (yellow) at lysine residue 36 (H3K36me3). In cells that do not include the alternative exon (pink) in the mRNA, the nucleosomes packaging this exonic DNA do not contain H3K36me3 (top). We propose that when RNA polymerase (brown ovals; spliceosome, orange circle) transcribes a gene with this chromatin configuration (with the pre-mRNA shown here in grey), it traverses the alternative exon rapidly, and this exon is not tethered to the RNA polymerase and, accordingly, sequence corresponding to this exon is not included in the mature mRNA (the splicing of exonic sequences is indicated by curved arrows). By contrast, when the nucleosome that is packaging the alternative exon contains H3K36me3, this slows the progress of the RNA polymerase, allowing it to capture the exon, resulting in the inclusion of sequence corresponding to this sequence in the mature mRNA (bottom). c, Kinetic model of mutually exclusive splicing of Dscam pre-mRNA. Mutually exclusive splicing of the sequence corresponding to the exon 6 cluster of the Dscam gene involves the formation of secondary structures in the RNA. These structures form between the docking site in the intronic sequence downstream of the sequence corresponding to exon 5 and a selector sequence located upstream of each sequence corresponding to an exon 6 variant. In addition, the hnRNP HRP36 binds to, and represses, all 48 exonic sequences in the cluster. Which exonic sequence is spliced seems to be a function of kinetic competition between the 48 potential docking-site–selector sequence structures and between SR proteins (not shown) and HRP36. On the left, the selector sequence upstream of the sequence corresponding to exon 6.3 in the pre-mRNA interacts with the docking site. HRP36 is then displaced from this exonic sequence, allowing an SR protein to bind in its place. This results in the inclusion of sequence corresponding to exon 6.3 in the mRNA. On the right, the formation of a secondary structure with a more distal sequence means that exon 6.47 is included in the mRNA instead.