The polypyrimidine tract binding protein (PTB, or hnRNP I)
contains four RNA-binding domains of the ribonucleoprotein fold
type (RRMs 1, 2, 3, and 4), and mediates the negative regulation
of alternative splicing through sequence-specific binding to
intronic splicing repressor elements. To assess the roles of
individual RRM domains in splicing repression, a neural-specific
splicing extract was used to screen for loss-of-function mutations
that fail to switch splicing from the neural to nonneural pathway.
These results show that three RRMs are sufficient for wild-type
RNA binding and splicing repression activity, provided that
RRM4 is intact. Surprisingly, the deletion of RRM4, or as few
as 12 RRM4 residues, effectively uncouples these functions.
Such an uncoupling phenotype is unique to RRM4, and suggests
a possible regulatory role for this domain either in mediating
specific RNA contacts, and/or contacts with putative splicing
corepressors. Evidence of a role for RRM4 in anchoring PTB binding
adjacent to the branch site is shown by mobility shift and RNA
footprinting assays.