The implications of alternative splicing in the ENCODE protein complement

Abstract

Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been suggested as one explanation for the discrepancy between the number of human genes and functional complexity. Here, we carry out a detailed study of the alternatively spliced gene products annotated in the ENCODE pilot project. We find that alternative splicing in human genes is more frequent than has commonly been suggested, and we demonstrate that many of the potential alternative gene products will have markedly different structure and function from their constitutively spliced counterparts. For the vast majority of these alternative isoforms, little evidence exists to suggest they have a role as functional proteins, and it seems unlikely that the spectrum of conventional enzymatic or structural functions can be substantially extended through alternative splicing.

Fig. 1.

Isoform distribution. (a) The number of isoforms per locus in the manually selected regions compared with the number of isoforms per locus in the regions selected by random stratified procedure. (b) The effect of splicing events on the protein sequence of alternative splice isoforms.

Fig. 2.

The potential effect of splicing on protein structure. Four splice isoforms mapped onto the nearest structural templates. Structures are colored in purple where the sequence of the splice isoform is missing. The deletions/substitutions will mean that the structures of these isoforms would require substantial reorganization from the parent structure. (a) Hemoglobin δ-subunit isoform 002 from locus AC104389.18 mapped onto PDB structure 1si4. (b) SET domain-containing protein 3, isoform 002 from locus AL132819.1 mapped onto structure 2h21. (c) Mitochondrial cysteine desulfurase isoform 006 from locus RP1-309K20.1 mapped onto structure 1p3w. (d). Eukaryotic initiation factor 6 isoform 005 from locus RP4-61404.1 mapped onto structure 1g62A. Biologically relevant heteroatoms are shown in space-filling format in b and c, and the heme in a is shown in stick format.

Fig. 3.

B serpins. Serpins are protease inhibitors that inactivate their targets after undergoing an irreversible conformational change. (a and b) Serpins exist in an inactivated form (a) that is regarded as being “stressed.” Cleavage of the 20-residue RSL region, missing in the structure but with the terminal ends shown in red, causes the RSL region to flip over and fit itself into one of the β-sheets (b, inserted RSL strand in red). This exposes the inhibitory region that inactivates the protease. (c–f) Four splice isoforms from different serpin loci mapped onto the structure of serpinB2 (1 × 7). The sections deleted/substituted in the isoforms are shown in purple. In each case, it appears that splicing is likely to cause the structure to fold in a substantially different fashion. Given that the complex structure of the inhibitor is vital to its unique function, it is not clear why so many apparently deleterious isoforms would be necessary.

Fig. 4.

The difficulty of modeling the structure of isoform IL-4d2. Splice isoform IL-4d2 (isoform 002 from locus AC004039.4) mapped onto structural template 1itl. The section coded for by the missing second exon is colored in dark gray. The cysteine bridges that stabilize the structure are shown in stick format. The missing exon would leave the flanking residues 30 Å apart, and the cysteine bridges mean that the structure has little room for reorganization. Solutions to the modeling problem would have to break the hydrophobic core of the four-helix bundle or break the cysteine bridges by realigning the helices.