SECISBP2 transcript expression and minigene-based in vivo splicing assay. (A) Relative expression levels of full-length and mitochondrial SBP2 transcripts in a cDNA panel of human tissues was analyzed by real-time PCR using transcript specific primers. Data was analyzed using the deltaCT method. (B) Schematic representation of the SECISBP2 minigene indicating exon/intron composition, location of primers used to amplify the minigene-expressed products, annealing site for antisense oligonucleotides (In2E3a ASO) and location of mutations introduced. (C) In vivo splicing pattern of SECISBP2 minigene constructs. RT-PCR of α-³²P-dCTP labeled products were amplified from C33A cells transfected with wild-type (WT), exon 2 ATG>CTG mutant (Ex2ATG>CTG) and exon 3 AG>AA mutant (Ex3AG>AA) minigenes. The exon combination as verified by sequencing is indicated on the left (products indicated by * are predicted only). Mutation at the exon 3a acceptor site AG>AA abolishes splicing between exon 2 and exon 3b as shown by the absence of exon 2–3b spliced product (lane 2). In2E3a ASO treatment almost completely abrogates exon 2–3a splicing and thus full-length SECISBP2 transcript and dramatically increases exon 2–3b splicing and mtSECISBP2 transcript levels (lane 4). Percentage of exon 3a skipping was calculated from the total value of exon 3a-included and exon 3a-skipped products. (D) Normal human fibroblast cells were treated with In2E3a ASO and levels of SECISBP2 and mtSECISBP2 were measured by real-time RT-PCR. In2E3a-treated cells show a 4-fold increase in mtSECISBP2 and 50% decrease in the full-length SECISBP2 transcript.

Localization of over-expressed mtSBP2-GFP and endogenous mtSBP2 isoform at the mitochondria. (A) The localization of GFP-tagged full-length (FLSBP2-GFP) and mitochondrial SBP2 isoforms (mtSBP2-GFP) in C33A cells was visualized by fluorescence microscopy. Mitochondria were stained with MitoTracker Red CMX-Ros. Single Z-layer deconvolution images show strong mitochondrial localization of mtSBP2-GFP (yellow) but not of FLSBP2-GFP. (B) Western blot of SBP2 isoforms expressed from cDNAs as GFP-fusion proteins. SBP2_Δ2-GFP and SBP2_Δ3a-GFP lack exons 2 and 3a respectively. mtSBP2-GFP cDNA begins at the ATG in exon 2. These proteins were resolved on a 6% SDS–PAGE gel, which yielded a higher level of separation compared to Figure 3. (C) High resolution single Z-layer deconvolution images show the localization of mtSBP2-GFP and endogenous mtSBP2 proteins at the mitochondria in C33A cells. 10% of cells display strong mitochondrial staining as shown by the yellow staining in the top panel enlargement, while in most cells only a few dots were detected within the mitochondria (middle panel). Immunofluorescence staining was performed with the anti-SBP2 antibody EC1. Enlargements show a similar punctated staining pattern along the mitochondria for both mtSBP2-GFP (bottom panel) and endogenous mtSBP2.

Schematic representation of human SECISBP2 transcript variants. (A) Genomic organisation of SECISBP2 showing the 17 exons and their sizes in base pairs (bp). Locations of the functional domains are indicated. (B) Alternatively spliced SECISBP2 transcript variants generated through different exon combinations are shown schematically. Arrowheads indicate translation start sites. Designation for the encoded protein isoforms, the corresponding Met start codon number [based on numbering in the full-length human SBP2 sequence (NP_076982)] and predicted molecular weights (MW) of isoforms are indicated to the right of each transcript. (C) An alternative reading frame used for translation of the mitochondrial SBP2 isoform (mtSBP2) initiates in exon 2 (indicated by arrow). The encoded mitochondrial targeting sequence is underlined.

Minigene-based expression of SBP2 isoforms. (A) The minigenes (described in the legend to Figure 2) were transfected into 293T cells and the proteins expressed were separated on 4–12% gradient gels and analyzed by western blotting with anti-GFP and anti-SBP2 antibodies. Western blots with the two antibodies show similar staining pattern and thus confirm the expression of several SBP2 isoforms initiating from alternative ATG codons. E3aSBP2-GFP, E3bSBP2-GFP and E5SBP2-GFP refer to isoforms initiating at ATG codons in exons 3a, 3b and 5 respectively. (B) Western blots of cells co-transfected with wild-type (WT) minigene and the In2E3a antisense oligonucleotides that promotes splicing of the mtSECISBP2 transcript show translation of the mtSBP2GFP isoform. Actin was used as a control for protein loading.

Coordinated transcriptional and translational regulation of SBP2 following UVA treatment. Transcriptional and translational response of full-length SBP2 (FLBSP2) and mitochondrial SBP2 (mtSBP2) following 50 kJ/m² of UVA irradiation was monitored in HeLa cells over 24 h. (A) Real-time RT-PCR using transcript specific primers shows a similar transcriptional induction of both transcripts in response to treatment. (B) Western blot using anti-SBP2 antibodies shows the translational response of endogenous SBP2 and HSP70 at different time points after UVA irradiation. Quantitation of FLSBP2 transcript and protein levels is presented in the graph below. Error bars represent the standard error of the mean (SEM). Transcript levels were normalized against GAPDH and protein levels were normalized against actin. (C) Western blots show the translational response of WT, Ex2ATG>CTG and Ex3AG>AA minigene-derived proteins to UVA treatment. HSP70 was used as a marker for UVA-induced stress and actin shows protein loading.