Article Figures & Data
Figures
- Fig. 1.
Expression of different survivin isoforms in human RCC cell lines. a, Northern blot hybridization revealed survivin expression in all RCC cell lines, irrespective of their histological subtypes (clear cell, chromophilic, and chromophobe types of RCC). Of note, a band of 1.3 kb indicated EPR-1 expression in clearCa-6. b, RT-PCR confirmed the results of Northern analysis showing survivin expression in all RCC cell lines as well as two additional fragments different in length from the survivin band. c, sequence analysis of the RT-PCR amplification products identified three different transcripts: The survivin transcript with four exons, survivin-2B with an additional exon (exon 2B) inserted between the exons 2 and 3, and survivin-ΔEx3, showing a loss of exon 3 as well as a frame shift with extension of the reading frame into the open reading frame of the 3′ untranslated region (arrows, PCR primer positions; flags, SD and SA sites; black boxes, the novel exon 2B; shaded boxes, the frame shift in exon 4 and the coding part of the former 3′ untranslated region). d, S1 analysis of the survivin-ΔEx3 transcript revealed the protected fragments of regular survivin (40 nucleotides) and alternatively spliced (80 nucleotides) survivin-ΔEx3 transcripts.
- Fig. 2.
Analysis of potential SD and SA sites between exon 2 and 3. a, computer-aided analysis of SD/SA sites was performed on the survivin genomic sequence to confirm the data obtained from RT-PCR and DNA sequencing. *, positions of potential SD and SA sites that correspond to the SD and SA sites used in the newly discovered survivin-2B splice variant. As a result, a part of the intron between exon 2 and 3 is retained as a cryptic exon, termed exon 2B (see Fig. 1c <$REFLINK> ). SD and SA sites were analyzed with the Signal program of the PC/Gene Package Ver. 6.60, according to the algorithm developed by Staden (13) . b, comparison of the SA and SD sites of exon 2B with the consensus sequences for SA and SD sites.
- Fig. 3.
Differential alterations of protein domains in the novel survivin splice variants. a, protein sequences were analyzed with the Prosite program of the PC/Gene package (21) . The BIR domain, which is known to be responsible for inhibition of certain caspases, was found to be modified in both alternative splice variants of survivin by computer analysis. Survivin-2B has acquired additional 23 amino acids encoded by the cryptic exon 2B. The novel domain introduces a potential N-glycosylation site as well as two potential N-myristoylation sites. Skipping of exon 3 in survivin-ΔEx3 results in a frame shift that causes a new COOH-terminal sequence with a potential N-myristoylation site. BIR′, modified BIR; PKC, protein kinase C phosphorylation site; N-Myr, N-myristoylation site; N-Glyc, N-glycosylation site; CK2, casein kinase 2 phosphorylation site; cAMP, cyclic AMP-dependent kinase phosphorylation site. b, multiple alignment, using Clustal W (22) and enhanced with Boxshade, of amino acid sequences of the three survivin variants. Darker shading is of residues that are highly conserved; lighter shading is of less well-conserved residues, and residues that are not shaded are not conserved.
- Fig. 4.
Differential antiapoptotic potential of the novel survivin splice variants. a, coding sequences of survivin and its two splice variants were ligated into the expression vector pEGFP-N3 and transfected into the human hepatoma cell line HepG2. Cell death was induced in transient transfectants by exposure to methotrexate (100 μg/ml). Survivin-ΔEx3 transfectants exhibited cell survival frequencies closely corresponding to that of survivin transfectants. In contrast, survivin-2B transfectants showed a marked reduction of cell survival. The transfection efficiency was ≥50% in all transfection experiments. The percentage of transfected cells was determined by microscopic evaluation of β-galactosidase expressing cells. Data are the means of at least three independent experiments; bars, SD. Statistical analysis was performed with Dunnett’s test. Of note, corresponding results were obtained using untagged pcDNA3.1-survivin/survivin-ΔEx3/survivin-2B constructs (data not shown). b, immunoblot analysis with both anti-GFP and anti-survivin antibodies revealed stable and comparable levels of expression for the indicated survivin variants in HepG2 transfectants. Of note, binding of anti-GFP antibody is independent of the survivin component of the fusion protein. Additional survivin-2B bands detected by the anti-GFP antibody might indicate posttranslational modifications of this survivin variant. The polyclonal anti-survivin antibody, which was generated against “regular” survivin, detected survivin splice variants with less intensities, as evident from comparison with α-tubulin controls.
Volume 59, Issue 24
