Fig 1.
The mouse Igf2—H19 locus and the human IGF2—H19 locus and genes.
A. Maps of the mouse Igf2 –H19 locus on chromosome 7 and the human IGF2 –H19 locus on chromosome 11p15.5 with chromosomal coordinates for key features listed. For IGF2/Igf2, exons are illustrated as boxes, with coding regions in red and noncoding in black, and introns as horizontal lines. Other genes are depicted as single boxes and include tyrosine hydroxylase (TH), insulin (Ins2, INS), noncoding RNA genes H19 and Nctc1, mitochondrial ribosomal protein L23 (Mrpl23, MRPL23), and troponin T3, fast skeletal type (Tnnt3, TNNT3). Horizontal arrows show the direction of gene transcription. Blue or yellow circles represent the mouse or human imprinting control region (ICR), respectively, which are located 5’ to H19 [15–17]; orange ovals indicate the 10 distal enhancers that were identified and functionally mapped in the mouse genome [40], and their 9 human homologues ([37] labeled as ‘conserved with distal enhancers’). A scale bar is indicated. B. Detailed view of mouse Igf2 and human IGF2 and both H19 genes, with exons as boxes (8 for Igf2, 10 for IGF2, 5 for mouse H19, and 6 for human H19), and introns and flanking DNA as horizontal lines. The letter ‘P’ indicates gene promoters (P0 and P1 –P3 for Igf2, P0 and P1 –P4 for IGF2, P for mouse H19, and P1 and P2 for human H19), and a scale bar is shown. For Igf2 and IGF2, non-coding exons are in black and coding exons are colored red. For H19, all exons are in black.
Fig 2.
Mouse Igf2 and human IGF2 mRNAs and proteins.
A, B. Depiction of the 4 major species of mouse Igf2 transcripts (A) and the 6 major types of human IGF2 mRNAs (B). The responsible promoters (p for mouse, P for human) are listed, as are the exons found in each transcript. The length of each mRNA is in nucleotides (nt). AN represents the polyadenylic acid tail found at the 3’ end of mRNAs. C. Depiction of mouse and human IGF2 protein precursors, showing the derivation of each segment from different Igf2 or IGF2 exons. Mature, 67-amino acid IGF2 is in blue; presumed and confirmed signal peptides (SP) are in black, and the 89-amino acid E peptide is in red. Amino acid is abbreviated as AA.
Fig 3.
Comparison of IGF2/Igf2 genes among mammals.
Schematics of human, gorilla, olive baboon, horse, and dog IGF2, and mouse, rat, guinea pig, elephant, and Tasmanian (Tas) devil Igf2 genes are shown. These are the genes for which the most information was extracted by searching genomic databases, as described in ‘Materials and methods’. Promoters (P) are labeled; the different terminology employed for mouse and rat promoters p1 to p3 (lower case) derives from genomic databases. All exons are indicated as boxes, with non-coding exons in black or gray and coding exons in red. The dark gray regions in exon 2 in human, gorilla, olive baboon, horse, and dog genes represent the additional part of the exon that is transcribed when P0 is active (exon 2 lg [large] in Table 1). Only the smaller black segment of exon 2 is transcribed when P1 is active (exon 2 in Table 1); it results from exon 1 splicing into exon 2 (see Fig 2B). The lighter gray portions of gorilla and horse exon 6 depict areas that have not been characterized because of poor quality DNA sequences (see Table 1). A question mark under horse IGF2 exon 5 indicates that the DNA sequence is found in a cDNA but could not be mapped to the horse genome, most likely because of poor quality genomic DNA sequence. The question mark adjacent to horse exon 4 indicates that no DNA sequence similar to human P2 could be mapped. Question marks under two Tasmanian devil Igf2 exons signify genomic DNA segments matching two Igf2 cDNAs that are not similar to IGF2/Igf2 noncoding exons in other mammalian species. A scale bar is also shown.
Table 1.
Percent nucleotide identity with human IGF2 exons¶.
Fig 4.
Detailed views of 10 mammalian H19 genes for which genomic data are relatively complete; exons are boxes, and introns and flanking DNA are horizontal lines. P1 and P2 depict the two gene promoters found in several primates. P denotes the gene promoter in other species. Bent arrows indicate different transcription start sites directed by human P2 and straight vertical arrows depict locations of alternative polyadenylation sites. The Tasmanian (Tas) devil H19 gene was mapped by similarity with the wallaby gene; the lighter gray portion of Tasmanian devil exon 5 depicts an area that could not be characterized because of poor quality DNA sequence (see Table 2). A scale bar is shown.
Table 2.
Percent nucleotide identity with human H19 exons¶.
Fig 5.
IGF2/Igf2 and H19 gene expression in mammals.
Data on IGF2/Igf2, H19, and control gene MRPS17/Mrps17 transcript expression in liver was obtained by screening RNA-sequencing libraries found in NCBI SRA (the libraries searched are listed in S1 Table, and the probes used in S2 Table). Results were graphed as hits identified per number of sequence reads in the library. A. IGF2/Igf2 mRNA levels were measured in human, cat, cow, dog, pig, rat, and Tasmanian (Tas) devil using probes containing coding exons that were equivalent to human exons 8 and 9 (see S2 Table). B. IGF2/Igf2 transcripts were assessed using probes containing each noncoding exon fused to the 5’ end of the first coding exon (the equivalent of human exon 8, see S2 Table). These results measure potential promoter use. C. H19 gene expression was evaluated in the same species as in A. D. MRPS17/Mrps17 (a potential control transcript) gene expression was assessed in the livers of the same species as in A.
Fig 6.
Amino acid sequences of IGF2 (67 or 68 amino acids) from different mammals are illustrated in single letter code. Dots depict identities, and differences among species are indicated. A dash depicts no residue. No IGF2 of this type could be identified in cat, megabat, wallaby, Tasmanian devil, or opossum, as indicated by the word ‘none’ (but see Fig 7).
Fig 7.
Mechanisms accounting for variant IGF2 proteins.
A. Amino acid sequences of a mammalian IGF2 variants with 70 or 71 amino acids are depicted in single letter code. The additional residues are underlined for human IGF2. Dots depict identities, and differences among species are indicated. A dash depicts no residue. B. The molecular basis for 70- or 71-amino acid IGF2 is a consequence of alternative splicing into the equivalent of human IGF2 exon 9, which adds an additional 9 nucleotides (in lower case and in red) to the 5’ end of the exon, and changes a serine codon into arginine-leucine-proline-glycine codons in IGF2 transcripts in human, gorilla, and macaque. In olive baboon, as based on a cDNA sequence deposited in GenBank, a different 5’ end of exon 9 has been proposed, which results in predicted serine-lysine-proline-glycine codons. This sequence cannot be identified at the 3’ end of IGF2 intron 8 in the olive baboon genome (as signified by *). In pig, horse, cat, dog, and megabat, different amino acids are found in the further COOH-terminal part of IGF2, as indicated in red. In wallaby and Tasmanian (Tas) devil, serine-leucine-proline-glycine comprise the variant amino acid quartet. This also may be true for opossum, but the relevant genomic DNA sequence is not available (thus **).
Table 3.
Amino acid identities with human IGF2 (%).
Fig 8.
Alignments of IGF2 signal peptides.
Amino acid sequences of IGF2 signal peptides from 19 mammals are shown in single letter code. A. 24-, 26-, or 28-residue IGF2 signal peptide 1 is found in all species except platypus. B. 80-, 85-, or 88-amino acid presumptive IGF2 signal peptide 2 can be detected only in human, gorilla, cow, horse, cat, dog, guinea pig, elephant, and platypus. The last 24 residues are identical to signal peptide 1 in human, gorilla, cow, horse, cat, dog, guinea pig, and elephant. For A and B, dots depict identities, dashes indicate no residues, and differences among species are shown. Note that in the platypus Igf2 gene, only a larger signal peptide is predicted that is unrelated in amino acid sequence to other the species depicted.
Fig 9.
Alignments of IGF2 E peptides.
Amino acid sequences of COOH-terminal IGF2 E peptides from 19 mammals are depicted in single letter code. Dots indicate identities, dashes depict no residues, and differences among species are shown. The IGF2 E domain comprises different lengths, ranging from 64 (cat) to 91 (opossum) amino acids.
Fig 10.
Comparison of IGF2/Igf2—H19 locus and genes in mammals.
Diagrams of human, gorilla, olive baboon, horse, and dog IGF2—H19, and mouse, rat, guinea pig, elephant, and Tasmanian (Tas) devil Igf2—H19 genes and loci are shown. For IGF2/Igf2 and H19, individual exons are indicated as boxes (coding regions are in red). Other genes are shown as single boxes, and include the following: tyrosine hydroxylase (TH/Th), insulin (INS/Ins2), noncoding RNA Nctc1 (mouse only), mitochondrial ribosomal protein L23 (MRPL23/Mrlp23), troponin T3, fast skeletal type (TNNT3/Tnnt3). A horizontal arrow indicates the direction of transcription for each gene. Yellow (primate) or aqua ovals (mouse and rat) depict the imprinting control region (ICR) 5’ to H19, and orange circles indicate homologues of the 10 distal enhancers that were identified and functionally mapped in the mouse genome [40], and identified by DNA sequence similarity in the other genes (see Table 4). A scale bar is also shown. Th is not illustrated on the maps for mouse or rat, as it is separated from Ins2 by ~226 Kb (mouse), and by ~222 Kb (rat).
Table 4.
Percent nucleotide identity with mouse Igf2-H19 locus enhancers¶.