Hypoxia-Induced Alternative Splicing in Endothelial Cells
Figure 4
Hypoxia-dependent alternative C-termini.
RT- and qRT-PCR analyses show changed expression of protein isoforms for itsn1 (A), larp6 (B) and max (C). Alternative splicing and polyadenylation lead to the expression of isoforms with different C-termini in istn1 (A) and larp6 (B). In the case of max (C) due to intron retention an alternative stop codon is introduced, also leading to a different C-terminus. Schemes of alternative splicing events: Introns are represented as black lines; exons as boxes; primer pairs (arrows) used to amplify different isoforms (i1, i2) or total amounts of the mRNAs (t) are indicated. White exons indicate isoform 1, which expression is changed under hypoxic conditions. Grey exons are common to both isoforms and primers spanning such regions were used to visualize changes in the total amount of the corresponding mRNA. In the case of larp6 only the first exon is common to both isoforms, therefore isoform 2 (black exon) was visualized instead of the total mRNA amount, using one common forward- and two isoform-specific reverse-primers. The primer pair recognizing the retained intron in the max mRNA could yield the same product with genomic DNA. To ensure sample purity and exclude falsification of the results by contamination with genomic DNA, we performed the same RT- (Figure S5) and qRT-PCR experiments with not reverse transcribed RNA (data not shown). These experiments yielded no product at all confirming the observed increase is due to intron retention. RT-PCR: N = normoxia, H = hypoxia, M = DNA size ladder. qRT-PCR: closed bars = normoxia, open bars = hypoxia. Expression is normalized to rplp0. Results from n = 2 (RT-PCR) and n = 3 (qRT-PCR larp6) or n = 6 (qRT-PCR itsn1 and max) experiments are shown. * = p-value<0.05, ** = p-value<0.01 (Student's t-test).