An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution

Abstract

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.

Figure 1

A. Solution structure of free IGF2 (yellow) and the free domain11^E4 (light green) and characteristic domain 11 β-barrel and four loops involved in IGF2 binding (AB, CD, FG, HI). B. The 24.2 kDa complex of domain11^E4 (blue) bound to IGF2 (yellow) solved by NMR. C. super-imposition of an ensemble of twenty domain 11^E4 low energy NMR structures showing the IGF2 binding pocket. The AB, CD, FG and HI loops are shown in green. D. backbone and surface representation of the IGF2 binding pocket highlighting a group of nine hydrophobes on domain11^E4, including the three foundation residues (L1626, L1636 and V1574) that support the binding pocket (green) and, in light blue, the hydrophobes that form the IGF2 binding pocket. The flexible AB and FG loops change conformation upon complex formation (purple arrows). E. hydrophobic binding residues on IGF2 (centre) and binding partners (dark blue, indicating favorable hydrophobic interactions) on domain11^E4 over the AB, CD, FG and HI loops (clockwise). F. the non-hydrophobic groups (charged, polar and hydrogen bond interactions) of IGF2 that interact with domain11^E4 are shown with matching complementarity in dark blue. Incorrect charge/ polar complementarity are shown in red. Yellow represents where either the acquisition of a charge or change in steric bulk of a residue cannot be assessed in the absence of a high-resolution structure.

Figure 2

A. high resolution NMR structures of domain 11 from chicken (red), echidna (orange), opossum (green) and human (magenta, PDB:2CNJ) and domain11^E4 (blue). (Table S2 provides a summary of structural statistics). Ensembles of the lowest twenty energy models are shown for each species. B. surface representations of the binding pocket of IGF2R-domain 11 and the acquisition of an increased hydrophobicity surrounding the IGF2 binding pocket (Movie S1). C. hydrophobic binding residues on IGF2 (centre) and binding partners (dark blue) on domain11^E4, human, opossum, echidna and chicken over the AB, CD, FG and HI loops. D. evolution of favorable charged, polar and hydrogen bond interactions between IGF2 and domain 11 species. (colors as in Fig. 1E, F).

Figure 3

A. Exonic splicing enhancer (ESE) densities at the CD-loop coding region of exon 34. The positions of predicted hexamer (Rescue-ESE) and Octamer (Chasin) ESEs are illustrated (Fig. S12). B. in vivo splicing of chicken, platypus, or hybrid exons 34 in chicken DF-1 cells (sequences of splice products provided in Fig. S13). Two complete splice products A and B (cryptic splice site, CS) are shown, with RT-PCR gel products showing expression of A product and suppression of B product by ESEs. FP, RP, and RT are forward, reverse and reverse transcriptase primers, respectively. C. mini-gene constructs and comparative enhancement of dsx mini-gene splicing in HeLa cell nuclear extracts by control (AAG₇ repeat) and ESEs (Fig. S14). D. phylogenetic context implies that IGF2-IGF2R binding site acquisition (light shade) occurred prior to the appearance of imprinting (dark shade), but was present within prototheria. Relative affinity increased in methatheria compared to prototheria, in keeping with a transition in binding site structure (CD loop). IGF2R is bi-allelically expressed in human, presumably because less selection pressure exists to maintain mono-allelic expression, and limits IGF2R imprinting to non-primate eutherians and metatheria. In terms of binding, favourable protein interactions are shown in blue, with incorrect charge and unpredictable complementarity shown in red and yellow, respectively. Silenced (imprinted) IGF2R allele is shown as ‘OFF’ compared to the expressed allele as ’ON’. For IGF2, the reciprocal imprinted alleles are present in both methatheria and eutheria, but absent in prototheria.