Figure 1.
Usage of four alternative poly(A) sites for 3′-end cleavage and polyadenylation of human HFE transcripts.
(A) The diagram represents the human HFE 3′ untranslated region comprising exons (Ex) six and seven. The length of the exons is shown in base pairs (bp). The position of native translation termination codon (STOP) is represented. Arrows above the diagram indicate the relative position of the four different forward primers (EX6F; EX6G; EX7G and EX7H) used in 3′-RACE experiments. The vertical black bars represent the poly(A) signals (numbered from 1 to 4) that were found to be used in the HFE mRNA 3′-end processing. Their sequence is also shown. Below, the thin lines represent the 3′-RACE products obtained by each primer (indicated on the left). The correspondence between each 3′-RACE product, its polyadenylation site and its length in bp is also represented. (B) Representative agarose gel electrophoresis showing 3′-RACE products from human liver total RNA, obtained by nested PCR using forward primers specified above each lane, the universal primer and the master mix provided by the BD SMART RACE cDNA Amplification Kit (BD Biosciences Clontech). The molecular weight marker (M) is the 1 kb DNA ladder (Invitrogen). (C) Schematic representation of the four human HFE 3′ untranslated regions identified and characterized by cloning and sequencing of the 3′-RACE products obtained from total RNA, isolated from duodenum, liver, heart, peripheral blood mononuclear cells (PBMCs), kidney, testis, spleen, small intestine and ovary. Again, vertical black bars represent the polyadenylation signal that is used in each isoform, with the corresponding sequence depicted and the distance from the poly(A) signal to the cleavage site given in nucleotides (nts). The size of each 3′ untranslated region is shown in nts, below each diagram. Each isoform is numbered from 1 to 4 according to the usage of the corresponding poly(A) signal, as depict above in A. (A)n represents the poly(A) tail. The table on the right shows the presence (✓) or absence (−) of each HFE alternative polyadenylation isoform in each tissue analyzed.
Figure 2.
Downregulating UPF1 from HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts which indicates that the physiological human HFE mRNA is a natural NMD-target.
HeLa cells were transiently transfected with synthetic small-interfering RNA (siRNA) duplexes directed to human UPF1 or to a non-endogenous target (Luciferase; Luc) used as control. Twenty-four (24 h), forty-eight (48 h) and seventy-two hours (72 h) after siRNA treatment, cells were harvested and protein and RNA were isolated. (A) Representative Western blot analysis of the HeLa and HepG2 cells extracts transfected with human UPF1 siRNA. Immunoblotting was performed using a human UPF1 specific antibody and a α-tubulin specific antibody to control for variations in protein loading. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved by densitometric analysis using ImageJ software. (B) Relative changes in HFE mRNA levels were analyzed by quantitative reverse transcription-coupled real-time PCR (RT-qPCR), normalized to the levels of endogenous G protein pathway suppressor 1 (GPS1) mRNA. For that, cDNA was synthesized from total RNA and then, each cDNA sample was used as template for qPCR, which was performed using the SYBR Green Master Mix (Applied Biosystems) and primers that specifically hybridize to the 5′-end of HFE exon seven. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed). Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. The double arrow represents the coordinates of the amplicon obtained in the qPCR. (C) Relative changes in HFE mRNA levels were quantified by RT-qPCR as in B but using primers that specifically hybridize to the 5′-end of the HFE exon six. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B. Below the histograms, there is a schematic representation of the human HFE mRNA showing its seven exons and the position of the polyadenylation [poly(A)] signals used in its 3′-end processing. The location of the initiation (AUG) and termination (STOP) codons is also represented. Arrows represent the position of primers used in the qPCR. (D) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or with a control Luciferase siRNA target. Twenty-four hours after siRNA treatment, cells were transfected with the β-globin reporter constructs (β39). Twenty-four hours post transfection, protein and RNA were isolated from the cells for analysis. Immunoblotting to confirm UPF1 knockdown was carried out with anti-UPF1 and with anti-α-tubulin antibodies (as a loading control). Identification of each band is on the right. The percentage (%) of UPF1 protein remaining expressed in the cells after siRNA treatment is indicated below each lane and was achieved as in A. (E) Relative β-globin mRNA levels in UPF1-depleted HeLa cells, normalized to the levels of puromycin resistance mRNA (plasmids carrying the reporter β-globin gene also contain the Puror gene), were determined by quantitative RT-qPCR and compared to the corresponding β39 mRNA levels under control conditions (Luc siRNA-treated HeLa cells) (defined as 1; arbitrary units). Using the same RNA samples, relative changes in HFE mRNA levels were also quantified by RT-qPCR, using experimental conditions as in B. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed as in B.
Figure 3.
Cycloheximide treatment of HeLa or HepG2 cells results in an upregulation of the endogenous HFE transcripts, confirming our previous conclusion that the physiological human HFE mRNA is a natural NMD-target.
HeLa or HepG2 cells were untreated (0 µmol/L) or treated with cycloheximide (200 µmol/L) during two hours. Then, cells were harvested and RNA was isolated. (A) Relative changes in HFE mRNA levels carrying polyadenylation at exon seven were analyzed by RT-qPCR, as previously in Figure 2B. Levels of HFE mRNA obtained after cellular cycloheximide treatment were compared to those obtained in untreated cells (defined as 1; arbitrary units). The histograms show the mean and standard deviations from three independent experiments. Below the histograms, there is a schematic representation of the human HFE mRNA (as in Figure 2). The double arrow represents the coordinates of the amplicon obtained in the qPCR. (B) Relative changes in total HFE mRNA levels were quantified by RT-qPCR as in A but using primers that specifically hybridize to the 5′-end of the HFE exon six, as shown in the diagram of the human HFE mRNA represented below the histograms.
Figure 4.
Contrary to the transcript corresponding to the minigene lacking intron 6, normal and nonsense-mutated human HFE transcripts are committed to NMD.
(A) Schematic representation of the studied human HFE minigenes: normal (WT), nonsense-mutated (Y138X), lacking intron six (Del_IVS6_WT) and nonsense-mutated lacking intron six minigene (Del_IVS6_Y138X). The position of the initiation (ATG) and termination (STOP) codons is represented. The name of each minigene is indicated above each diagram. (B) Representative Western blot analysis of HeLa cells extracts transfected with human UPF1 siRNA or a control siRNA target (siRNA Luciferase). Twenty-four hours after siRNA treatment, cells were co-transfected with the plasmids encoding the above referred minigenes and with a second dose of siRNAs (UPF or Luciferase). Twenty-four hours later, cells were harvested for protein and RNA. Immunoblotting was performed using a human UPF1 specific antibody and an α-tubulin specific antibody to control for variations in protein loading. (C) HFE mRNA quantification was performed by RT-qPCR using primers specific for the heterologous 5′ UTR common to all transfected genes. Neomycin resistance transcript was used as a normalization control. Quantification of the transcript levels was performed by the absolute quantification method. Levels of HFE mRNA obtained after cellular UPF1 siRNA treatment were compared to those obtained after Luciferase siRNA treatment at the same conditions (defined as 1; arbitrary units). The histogram shows the mean and standard deviations from three independent experiments, corresponding to three independent transfections. Statistical analysis was performed using Student's t test (unpaired, two tailed).
Table 1.
DNA oligonucleotides used in the current work.