Figure 1.
Identification of transcription initiation site of IGF2BP2 by 5′RLM-RACE.
Total RNA from human HEK293 fibroblasts, mouse 3T3-L1 preadipocytes and rat placenta was used as templates for the RACE experiment. (A) Schematic representation of 5′RLM-RACE PCR. Two forward primers recognizing the adapter sequence, and two reverse primers against exon 6 of human/mouse/rat IGF2BP2 are shown. The inner primer is 467 nt downstream of the canonical translational start site in mouse and rat, and 473 nt downstream in human. (B) Agarose gel electrophoresis of nested PCR reaction products. Molecular size markers (base pairs) are indicated on the left. The correct product is indicated by a filled triangle, and the non-specific product is indicated by an open triangle. M: marker, h: human, m: mouse, r: rat, −ve: PCR negative control using water as template, −TAP: negative control using RNAs that was not treated with TAP, therefore could not ligate to the RNA adapter. (C) Sequence alignment of human, mouse and rat 5′UTR as identified by 5′RLM-RACE. The transcription start sites are in bold and underlined. The canonical translation initiation site is shown in red. The position of the most 5′ transcription start site relative to the translation initiation site is indicated at the end of the sequences.
Figure 2.
Detection of a novel IGF2BP2 isoform in vivo.
A. Expression of endogenous IGF2BP1/2/3 expression in human, rat and mouse. Samples (10 µg of total protein) from human HEK293 cells, rat placenta, mouse placenta, mouse embryo E18 (mE18) and 3T3-L1 cells were resolved by 10% SDS-PAGE and analysed by Western blot using an antibody against an internal fragment of human IGF2BP2 (Abnova) (panel 1) and antibodies against the C-terminus of mouse IGF2BP2/1/3 (CRB Ltd) (panels 2, 3 and 4). B. Knockdown and overexpression of IGF2BP2 in 3T3-L1 preadipocytes using retrovirus. 3T3-L1 cells were treated with control scrambled shRNA (Scr) or two independent shRNA constructs targeting endogenous IGF2BP2 (sh1-BP2 and sh2-BP2) (left panels). An empty control vector (pBabe) and a vector containing IGF2BP2 cDNA sequence (BP2/pBabe) were also introduced into 3T3-L1 cells to overexpress the protein (right panels). Samples (10 µg of total protein) from human HEK293 cells, rat placenta, mouse placenta, mouse embryo E18 (mE18) and 3T3-L1 cells were resolved by 10% SDS-PAGE and analysed by Western blot using antibodies against the C-terminus of mouse IGF2BP2/1/3 (CRB Ltd) (panels 1, 2 and 3). In both experiments untreated 3T3-L1 cells are in the left-most lane. Positions of molecular weight markers are indicated.
Figure 3.
Tissue distribution of IGF2BP2 isoforms.
Expression of IGF2BP2 was analysed by Western blotting in tissues from 3-day old rat (A) and E19 mouse embryo (B). Total cellular proteins in lysates prepared from the tissues indicated (25 µg/lane) were resolved by 10% SDS-PAGE and blots were probed with antibody to IGF2BP2 C-terminal peptide (CRB Ltd). Positions of molecular weight markers are indicated to the right.
Figure 4.
Identification of Met69 as the putative alternative translation initiation site.
A. Schematic representation of IGF2BP2 and locations of the canonical initiation site at Met1/2 (solid arrow) and the putative alternative translational initiation site at Met69 (broken arrow). B. Protein sequence alignment of the 5′ region of IGF2BP2 in human, chimpanzee, cow, mouse and rat,. Two putative translational initiation sites are shown by arrows with solid and broken lines, respectively. C. Nucleic acid sequence alignment of IGF2BP1/2/3 translational initiation sites and the surrounding sequences. Bases that match the consensus are in upper case, while those that do not match the consensus are in lower case. The start codons are in red, and the most two important positions in the Kozak consensus are in bold.
Figure 5.
The p58 isoform of IGF2BP2 is generated by leaky ribosomal scanning.
A. A schematic representation of mIGF2BP2 mutants. Only the sequences surrounding the canonical translation initiation site and the internal Met69 are shown. The two putative translation initiation sites are in red. Cds, wild-type coding sequence of IGF2BP2; UTR, wild-type IGF2BP2 with 34 nt 5′UTR and 336 nt 3′UTR sequences included; Kozak1/2, wild-type IGF2BP2 with added Kozak sequence upstream of the canonical starting codon, 1/2Ile, the first and second ATG codon was mutated to ATC; 69Ile, the internal Met69 codon was mutated to ATC, mutKozak69, the Kozak consensus sequence flanking the internal Met69 was mutated; Δ5′, the first 66 codons including the first and second ATG codons were deleted. B. Protein analysis of mouse IGF2BP2 mutants expressed in 3T3-L1 preadipocytes (upper panels) and NIH-3T3 fibroblasts (lower panels) using retrovirus vectors. Untreated cells, and cells transfected with the empty vector pBabe, were used as controls. Cellular proteins (10 µg/lane) were resolved by 10% SDS-PAGE and blots were probed with antibody to IGF2BP2 C-terminal peptide (CRB Ltd) and with antibody to the p85 subunit of PI3-kinase as a loading control.
Figure 6.
The p58 isoform of IGF2BP2 is not generated by protease cleavage.
A. A schematic representation of the frameshift mutation. A single nucleotide deletion at position 198 resulted in a premature stop codon in the ORF starting at the double Met but did not affect the downstream ORF starting from the internal Met69. A Kozak sequence was also added upstream of the canonical double Met. B. Western blot analysis of the frameshift construct expressed in 3T3-L1 preadipocytes. Empty vector (pBabe) or the frameshift construct (Δnt198) was introduced into the cells using retrovirus. Cellular proteins (10 µg/lane) were resolved by 10% SDS-PAGE and blots were probed with antibody to IGF2BP2 C-terminal peptide (CRB Ltd) and with antibody to the p85 subunit of PI3-kinase as a loading control. Positions of molecular weight markers are indicated to the right.
Table 1.
Sequences of oligonucleotides and primers.