Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Logo for

Science 338 (6111): 1209-1213

Copyright © 2012 by the American Association for the Advancement of Science

An Exon Splice Enhancer Primes IGF2:IGF2R Binding Site Structure and Function Evolution

Christopher Williams1,*, Hans-Jürgen Hoppe2,*, Dellel Rezgui2, Madeleine Strickland1, Briony E. Forbes3, Frank Grutzner3, Susana Frago2, Rosamund Z. Ellis1, Pakorn Wattana-Amorn1, Stuart N. Prince2, Oliver J. Zaccheo2, Catherine M. Nolan4, Andrew J. Mungall5, E. Yvonne Jones6, Matthew P. Crump1,{dagger}, and A. Bassim Hassan2,{dagger}

1 Department of Organic and Biological Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.
2 Cancer Research UK Tumour Growth Control Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK.
3 School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
4 School of Biology and Environmental Science, University College Dublin, Belfield, Dublin, Ireland.
5 Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, Canada.
6 Cancer Research UK Receptor Structure Research Group, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.

Figure 1
View larger version (50K):
[in this window]
[in a new window]

Fig. 1. (A) Solution structure of free IGF2 (yellow), the free domain 11E4 (light green), the characteristic domain 11 β barrel, and four loops involved in IGF2 binding (AB, CD, FG, HI). (B) The 24.2-kD complex of domain 11E4 (blue) bound to IGF2 (yellow) solved by NMR. (C) Superimposition of an ensemble of 20 domain 11E4 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 domain 11E4, including the three foundation residues (L1626, L1636, and V1574) (15) 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 (center) and binding partners (dark blue, indicating favorable hydrophobic interactions) on domain 11E4 over the AB, CD, FG, and HI loops (clockwise). (F) The nonhydrophobic groups (charged, polar, and hydrogen bond interactions) of IGF2 that interact with domain 11E4 are shown with matching complementarity in dark blue. Incorrect charge and 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
View larger version (67K):
[in this window]
[in a new window]

Fig. 2. (A) High-resolution NMR structures of domain 11 from chicken (red), echidna (orange), opossum (green), and human (magenta, PDB:2CNJ) and domain 11E4 (blue). (table S2 provides a summary of structural statistics.) Ensembles of the lowest 20 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) (15). (C) Hydrophobic binding residues on IGF2 (center) and binding partners (dark blue) on domain 11E4, 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. 1, E and F.


Figure 3
View larger version (31K):
[in this window]
[in a new window]

Fig. 3. (A) 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) Minigene constructs and comparative enhancement of dsx minigene 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 before the appearance of imprinting (dark shade) but was present within prototheria. Relative affinity increased in methatheria compared with prototheria, in keeping with a transition in binding site structure (CD loop). IGF2R is biallelically expressed in human, presumably because less selection pressure exists to maintain monoallelic expression, and limits IGF2R imprinting to nonprimate eutherians and metatheria. In terms of binding, favorable 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 with the expressed allele as ON. For IGF2, the reciprocal imprinted alleles are present in both methatheria and eutheria but absent in prototheria.


To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882