P2X6 subunit interacts with SF3A1 and Spectrin α2 in nuclear extracts from hippocampus of 8 month-old mice and alters splicing efficiency <i>in vitro</i>.

<p>(<b>a</b>) Western blot of subcellular fractions from 8 month-old mice hippocampus. P2X6 subunit was located in both the cytosolic (cyt) and the nuclear fraction (nuc), as identified by localization with either glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or NeuN (neuronal nuclei marker)/histone 2b (H2b), respectively. (<b>b</b>) Nuclear extracts immunoprecipitated with anti-P2X6 antibody were resolved in a 2D electrophoresis gel and selected spots were isolated, processed and identified by MALDI/TOF (n = 3 mice). (<b>c-f</b>) Nuclear extracts immunoprecipitated with either IgG or anti-P2X6 antibodies were analysed by immunoblotting with antibodies against SF3A1 (<b>c</b>) and spectrin α-2(<b>d</b>). Nuclear extracts were also immunoprecipitated with spectrin α-2 (<b>e</b>) or SF3A1 (<b>f</b>) antibodies and immunoblotted with anti-P2X6 antibody. (<b>g</b>) Scheme shows four DNA constructions prepared for splicing activity quantitation and their respective protein translations. (<b>h</b>) Splicing inhibitor isoginkgetin (IGK) was used to validate the molecular tools employed to quantify the splicing efficiency. As expected TPI I firefly luciferase-dependent luminescence decreases and TPI II firefly luciferase-dependent luminescence increases when splicing is impaired (mean±s.e.m., n = 5 independent experiments, * P<0.05 unpaired Student’s t-test). (<b>i</b>) Overexpression of full-length P2X6 subunit reduces splicing activity in neuroblastoma cells compared to either control (GCL) or N14P2X6 transfected cells. The inhibition is similar to those produced by IGK (mean±s.e.m normalized values, n = 3 independent experiments, * P<0.05 unpaired Student’s t-test, ns P>0,05 unpaired Student’s t-test). (<b>j</b>) Western blot of subcellular fractions from neuroblastoma cells transfected with YFP, P2X6-YFP or N14P2X6-YFP, showing an increase of SF3A1 in the nuclear fraction only in P2X6-YFP transfected cells. (<b>k</b>) Western blot of hippocampal nuclear fractions from 8 month-old mice showing an increase of SF3A1 in the nucleus (n = 4 mice, *P<0.05 unpaired Student’s t-test). (<b>l</b>) Developing mice (E14.5) were <i>in utero</i> electroporated with YFP or P2X6-YFP and harvested at P0. Brain sections were labeled with antibodies against GFP and SF3A1, showing an increase of SF3A1 in the nucleus of transfected neurons. Arrows heads indicate transfected cells with high SF3A1 expression and full arrows show evident dotted pattern colocalization of SF3A1 and YFP. Scale bar 25 μm. Enlarged view scale 5 μm.