In this study, we demonstrate the ability of the
polypyrimidine tract binding protein PTB to function as
a coordinator of splicing regulation for a trio of neuron-specific
exons that are subject to developmental splicing changes
in the rat cerebellum. Three neuron-specific exons that
show positive regulation are derived from the GABAA
receptor γ2 subunit 24 nucleotide exon, clathrin light
chain B exon EN, and N-methyl-D-aspartate receptor
NR1 subunit exon 5 pre-mRNAs. The functional activity of
splicing repressor signals located in the 3′ splice
site regions adjacent to the neural exons is shown using
an alternative splicing switch assay, in which these short
RNA sequences function in trans to switch splicing
to the neural pathway in HeLa splicing reactions. Parallel
UV crosslinking/competition assays demonstrate selective
binding of PTB in comparison to substantially lower binding
at adjacent, nonneural 3′ splice sites. Substantially
lower PTB binding and splicing switch activity is also
observed for the 3′ splice site of NMDA exon 21,
which is subject to negative regulation in cerebellum tissue
in the same time frame. In splicing active neural extracts,
the balance of control shifts to positive regulation, and
this shift correlates with a PTB status that is predominantly
the neural form. In this context, the addition of recombinant
PTB is sufficient to switch splicing to the nonneural pathway.
The neural extracts also reveal specific binding of the
CUG triplet repeat binding protein to a subset of regulatory
3′ splice site regions. These interactions may interfere
with PTB function or modulate splicing levels in a substrate-specific
manner within neural tissue. Together these results strengthen
the evidence that PTB is a splicing regulator with multiple
targets and demonstrate its ability to discriminate among
neural and nonneural substrates. Thus, a variety of mechanisms
that counterbalance the splicing repressor function of
PTB in neural tissue are capable of mediating developmental
splicing control. Altered expression of PTB isoforms during
cerebellar development, as documented by Western blot analysis,
is proposed to be a contributing mechanism.