Figure 1.
HMOX1 transcription start site usage in human tissues.
Graphical representation of the gene HMOX1 on human chromosome 22q12 and the two expressed sequence tags BE407102 and DA903962. Boxes represent exonic regions and constant lines intronic sequence. 5’ RACE was performed on human cDNA tissue panels. RACE primers are indicated by white arrows. Transcription start sites (TSSs) represent the first nucleotide of the transcripts. TSS positions are given as nucleotides relative to the annotated TSS (GRCh37/hg19, chr22:35,777,060) at zero (black arrow). The canonical translation start codon in exon 1 is indicated by ATG.
Figure 2.
Re-evaluated 5’ region of the human gene HMOX1.
(A) Exon-intron structure of HMOX1. Exons are represented by boxes, whereas white boxes represent novel exonic region. The two independent transcription initiation sites are indicated by black arrows. ATG is the translation start codon of the HO-1 coding region. uATG and downstream STP (stop codon) define the upstream open reading frame (uORF). (B) Sequence of the HMOX1 5’ region. Exons are represented by capitalized letters and the annotated first exon is highlighted by a grey box. Bolt italic letters correspond to the SNP rs2071745 and the (GT)n microsatellite. The first nucleotide of the common transcription start sites are boxed in black. Alternative splice acceptor sites (AG1-AG4) at exon 1 are numbered above. Translation start codons are in bold letters and stop codons are underlined. The canonical ATG is marked by an asterisk.
Figure 3.
Polymorphism-dependent alternative splicing at the HMOX1 5’ region.
(A) Schematic representation of the HMOX1 minigene and expressed splice-isoforms. Boxes represent exonic regions and constant lines intronic sequence. Black boxes are exonic sequences provided by the pcDNA3.3 plasmid. The CMV promoter is represented by a triangle. Given numbers correspond to nucleotide length. Primers for RT-PCR are indicated by white arrows. (B) The amount of HMOX1 minigene splice-isoforms found in HEK293-EBNA and HepG2 cells are given as percent fractions (means of n=3 ± standard deviation). The significance level for the differences in the splice-isoform profiles between the six haplotypes was calculated with the G-test (**p<0.001). (C) Changes in HMOX1 minigene (N30/A) percent splice-isoform fractions in HepG2 cells treated with 10µM hemin for 20 hours. Data are plotted as means (n=3) ± standard deviation. Statistical significance was calculated with respect to the control using t-test (*p<0.05).
Figure 4.
Alternative 5’ UTR splicing effects HO-1 translation efficiency.
(A) Schematic representation of the HMOX1 5’ UTR luciferase gene reporter constructs. Boxes represent transcribed regions included in reporter constructs. pGL3 promoter vector contains SV40 promoter (white triangle), transcription start site (black arrow) and firefly luciferase gene (black box). HMOX1 sequence context includes translation initiation codon (ATG) in exon 1 (canonical ATG) and in exon 2 as well as upstream open reading frame features uATG and STP (stop codon). (B) Relative luciferase units (RLUs) are given either for HepG2 or HEK293-EBNA cells transfected with the luciferase gene reporter constructs. Values are background subtracted and normalized to non-modified pGL3 promoter vector (pGL3-LUC). Data are plotted as means (n=9) ± standard deviation. Significance levels were calculated versus Exon1-LUC construct for each cell line independently using the Dunnett’s test (* p<0.05).