PQ treatment or nuclear SRPK2 affect splice site selection of the E1A minigene transcript.

<p>A. Schematic diagram illustrating the structure of the construct and the splicing pattern of the E1A minigene reporter. Arrows mark the position of the primers used for PCR analysis. The alternative 5′ splice site and splicing events that generate the different mRNA variants are indicated. B. Representative agarose gel of the splicing assay. SH-SY5Y cells were transiently transfected with the E1A reporter plasmid. 24 h after transfection cells were treated PQ. Total RNA was then isolated and the alternative splicing pattern of the E1A transcripts was determined by RT-PCR. The treatment with PQ induced an increase of the production of the 9 S transcript variant with respect to the other isoforms. C. Densitometric analysis of the E1A splicing products (mean ± S.E., n = 3) in cells treated with vehicle or with PQ. *** indicates p<0.001, and * indicates D. p<0.05, compared with control by paired, two-tailed Student's t test. E. Western blot analysis of the expression level of HA-tagged wild type and the S581D mutant in untreated and in PQ-treated SH-SY5Y cells. Blots were probed with an anti-HA antibody. Actin was used as loading control. F. SH-SY5Y cells were transiently transfected with the E1A splicing reporter minigene alone or together with expression plasmids coding for HA-tagged wild type SRPK2 or with the S581D mutant. RNA and protein fractions were simultaneously prepared. The alternative splicing pattern of the E1A transcripts was determined by RT-PCR. G. Densitometric analysis of the splicing products (mean ± S.E., n = 3) in untreated and PQ-treated cells. The relative levels of 13 S, 12 S, and 9 S mRNAs were quantitated as described in Materials and Methods. *** indicates p<0.001, * * indicates p<0.01, by one-way ANOVA and Dunnett's post test.