Figure 4. hnRNP A2/B1-dependent AS in the D-amino acid oxidase (DAO) gene results in reduced DAO protein expression and enzymatic activity.

(A) Gene models of the long and short mRNA isoforms produced by AS of the hnRNP A2/B1-dependent cassette exon (in white) in the Dao pre-mRNA. Unaffected exons are shown as black boxes. Introns and UTRs are shown as thin boxes and lines, respectively. The lengths of the predicted protein products are indicated. Amino acid (AA).

(B) The transcript levels of Dao mRNA in control and hnRNP A2/B1 depleted mouse spinal cord (n = 4 mice per group) determined by qPCR. Tbp was used as reference. Error bars are SEM computed with four replicates. The mean transcript level for the control mice is normalized to 1.

(C) Gel-like image of AS of the Dao cassette exon in mice, determined by RT-PCR, shows exon exclusion upon hnRNP A2/B1 depletion.

(D) Western blotting shows a reduction in DAO protein levels to ~70% of non-target control levels in mice treated with hnRNP A2/B1 targeting ASO. Actin was the loading control.

(E) X-ray crystallography structure of human DAO (left; Protein Database ID 2E48, Kawazoe et al. (2006)) and the predicted structure of the short isoform (right). The arrows denote two alpha helices and three beta sheets that are absent in the predicted shorter isoform.

(F) Schematic of constructs used to express DAO long and short isoforms in stable cell lines. Western blotting shows reduced protein levels in clones expressing the short isoform. Tubulin was the loading control.

(G) No difference in transcript level of Dao mRNA from the clones shown in (F) determined by qPCR. Tbp was used as a reference transcript. Error bars are SEM computed with four replicates per condition.

(H) DAO activity in the clones shown in (F). Bars represent the ratio of substrate to non-substrate dependent activity. Error bars are SEM computed with four replicates per condition.

(I) Western blotting of DAO protein translated in rabbit reticulocyte lysate reveals similar levels in cells expressing either isoform.

(J) DAO activity in the lysates shown in (J). The wedge below the bars denotes serial two-fold dilutions of the lysates.

(K) Depletion of hnRNP A2/B1 in the human U-251 glioblastoma cell line. Three different targeting shRNAs and one non-targeting control shRNA plasmids were used in triplicates. Splicing-sensitive RT-PCR (top) for DAO shows that under conditions where the short DAO isoform cannot be detected in the NTC-shRNA treated samples, the short DAO isoform was consistently detected in samples treated with the two most effective shRNA constructs. hnRNP A2/B1 depletion was verified by western blotting (bottom). Tubulin was the loading control.