Copper-binding proteins and exonic splicing enhancers and silencers.

Eukaryotic DNA codes not only for proteins but contains a wealth of information required for accurate splicing of messenger RNA precursors and inclusion of constitutively or alternatively spliced exons in mature transcripts. This "auxiliary" splicing code has been characterized as exonic splicing enhancers and silencers (ESE and ESS). The exact interplay between protein and splicing codes is, however, poorly understood. Here, we show that exons encoding copper-coordinating amino acids in human cuproproteins lack ESEs and/or have an excess of ESSs, yet RNA sequencing and expressed sequence tags data show that they are more efficiently included in mature transcripts by the splicing machinery than average exons. Their largely constitutive inclusion in messenger RNA is facilitated by stronger splice sites, including polypyrimidine tracts, consistent with an important role of the surrounding intron architecture in ensuring high expression of metal-binding residues during evolution. ESE/ESS profiles of codons and entire exons that code for copper-coordinating residues were very similar to those encoding residues that coordinate zinc but markedly different from those that coordinate calcium. Together, these results reveal how the traditional and auxiliary splicing motifs responded to constraints of metal coordination in proteins.

Exonic splicing code and coordination of divalent metals in proteins.

Exonic sequences contain both protein-coding and RNA splicing information but the interplay of the protein and splicing code is complex and poorly understood. Here, we have studied traditional and auxiliary splicing codes of human exons that encode residues coordinating two essential divalent metals at the opposite ends of the Irving-Williams series, a universal order of relative stabilities of metal-organic complexes. We show that exons encoding Zn2+-coordinating amino acids are supported much less by the auxiliary splicing motifs than exons coordinating Ca2+. The handicap of the former is compensated by stronger splice sites and uridine-richer polypyrimidine tracts, except for position -3 relative to 3' splice junctions. However, both Ca2+ and Zn2+ exons exhibit close-to-constitutive splicing in multiple tissues, consistent with their critical importance for metalloprotein function and a relatively small fraction of expendable, alternatively spliced exons. These results indicate that constraints imposed by metal coordination spheres on RNA splicing have been efficiently overcome by the plasticity of exon-intron architecture to ensure adequate metalloprotein expression.

Exonic splicing code and protein binding sites for calcium.

Auxilliary splicing sequences in exons, known as enhancers (ESEs) and silencers (ESSs), have been subject to strong selection pressures at the RNA and protein level. The protein component of this splicing code is substantial, recently estimated at ∼50% of the total information within ESEs, but remains poorly understood. The ESE/ESS profiles were previously associated with the Irving-Williams (I-W) stability series for divalent metals, suggesting that the ESE/ESS evolution was shaped by metal binding sites. Here, we have examined splicing activities of exonic sequences that encode protein binding sites for Ca2+, a weak binder in the I-W affinity order. We found that predicted exon inclusion levels for the EF-hand motifs and for Ca2+-binding residues in nonEF-hand proteins were higher than for average exons. For canonical EF-hands, the increase was centred on the EF-hand chelation loop and, in particular, on Ca2+-coordinating residues, with a 1>12>3∼5>9 hierarchy in the 12-codon loop consensus and usage bias at codons 1 and 12. The same hierarchy but a lower increase was observed for noncanonical EF-hands, except for S100 proteins. EF-hand loops preferentially accumulated exon splits in two clusters, one located in their N-terminal halves and the other around codon 12. Using splicing assays and published crosslinking and immunoprecipitation data, we identify candidate trans-acting factors that preferentially bind conserved GA-rich motifs encoding negatively charged amino acids in the loops. Together, these data provide evidence for the high capacity of codons for Ca2+-coordinating residues to be retained in mature transcripts, facilitating their exon-level expansion during eukaryotic evolution.