Exonic splicing code and protein binding sites for calcium

Abstract

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.

Selection of Olduvai Domains during Evolution: A Role for Primate-Specific Splicing Super-Enhancer and RNA Guanine Quadruplex in Bipartite NBPF Exons

“…The scores, which are expressed here as ESE/ESSseq ratios, estimate the strength of exon selection and predict the exon–intron architecture by specifying a gradient of exon and intron definitions in primary transcripts [ 8 , 13 ]. In addition, the ESE/ESS and their scores carry a significant protein [ 14 ] and protein domain-related [ 9 , 15 ] information.…”

“…The ESE/ESSseq profiles in the first exon of the HLS1 doublets were, thus, distinct from the first exon of a CON sub-type, where most splice-enhancing activity was found in the vicinity of the 3′ splice site ( cf. Figure S9 in [ 9 ]).…”

“…The 5′ part of the first exon encoding CON1 in NBPF genes resembles a trinucleotide repeat (Figure S9 in [ 9 ]). Trinucleotide-containing RNAs and other microsatellites can form assemblies of intracellular RNA aggregates that cause more than 30 genetic disorders by sequestrating RNA-binding proteins, leading to downstream changes in alternative splicing and spliceopathies [ 29 ].…”

“…For example, a GC-rich microsatellite expansion in the first CNBP intron in type 2 myotonic dystrophy can trigger intron retention [ 30 ]. Both the trinucleotide-like exonic segment [ 9 ] and the more recent pG4-led expansions of HLS1 sub-types [ 6 ] showed strong purine enrichment ( Figure 1 A). A nearly exclusive purine composition of pG4 is likely to diminish intramolecular Watson–Crick base-pairing in these regions and increase their RNA single-strandedness in the absence of non-canonical structures.…”

“…Finally, an underappreciated aspect of G4 biology is the intercalation of monovalent and divalent metal ions into the centre of or between G4 tetrads, stabilizing them in a metal-specific manner and enhancing their base-stacking interactions [ 35 , 36 , 37 ]. The evolution of the ESE and ESS was tailored by binding sites of divalent metals, such as Ca 2+ , reflecting their position in the Irving–Williams stability series [ 9 , 15 ]. Both mono- and divalent metal ions are pivotal for G4 stability gradients, which have been linked to their ionic radius, hydration energy and binding strength toward the guanine O 6 [ 37 ].…”