Cell- and stage-specific splicing events resolved in specialized neurons of the rat cerebellum.
Tissue and stage-specific pre-mRNA splicing events are important for posttranscriptional gene control, yet the diversity of such regulatory pathways has been largely unexplored at the single-cell level. Here we use a less conventional approach, which combines the whole-cell patch clamp method and the reverse transcriptase-polymerase chain reaction, to examine five neuron-specific splicing events in individual Purkinje neurons during postnatal development in live slices of rat cerebellum. Within the dimensions of the slice, the neurons sampled in this manner remain connected in their natural circuits and express multiple neuron-specific mRNAs, unlike established cell lines. In contrast to invariant splicing of control mRNAs, significant changes in splicing regulation during development are displayed by regulated exons of the GABA(A) receptor gamma2 subunit, clathrin light chain B, neural cell adhesion molecule, and N-methyl-D-aspartate receptor R1 mRNAs. Whereas two of the neuron-specific exons are regulated in parallel in Purkinje neurons, these same substrates are regulated differentially in cerebellar Granule neurons during the same course of development. These results illustrate how two types of specialized neurons contribute to splicing regulation in the natural environment of the complex tissue. In addition, these results provide a larger view of splicing regulation, favoring models in which cell-specific machineries operate in a more selective, rather than widespread manner, in these neuronal cell types.
Wang
Z
Z
Howard Hughes Medical Institute and Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Grabowski
P J
PJ
D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D066298:In Vitro Techniques; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D011689:Purkinje Cells / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results
D012333:RNA, Messenger