High-affinity binding sites for the hnRNP A1 protein stimulate
the use of a distal 5′ splice site in mammalian pre-mRNAs.
Notably, strong A1-mediated shifts in splice site selection
are not accompanied by equivalent changes in the assembly of
U1 snRNP-containing complexes on competing 5′ splice sites.
To explain the above results, we have proposed that an interaction
between hnRNP A1 molecules bound to high-affinity sites loops
out the internal 5′ splice site. Here, we present additional
evidence in support of the looping out model. First, replacing
A1 binding sites with sequences that can generate a loop through
RNA duplex formation activates distal 5′ splice site usage
in an equivalent manner. Second, increasing the distance between
the internal 5′ splice site and flanking A1 binding sites
does not compromise activation of the distal 5′ splice
site. Similar results were obtained with pre-mRNAs carrying
inverted repeats. Using a pre-mRNA containing only one 5′
splice site, we show that splicing is repressed when flanked
by two high-affinity A1 binding sites or by inverted repeats,
and that inactivation of the internal 5′ splice site is
sufficient to elicit a strong increase in the use of the distal
donor site. Our results are consistent with the view that the
binding of A1 to high-affinity sites promotes loop formation,
an event that would repress the internal 5′ splice site
and lead to distal 5′ splice site activation.