The SR proteins are essential metazoan pre-mRNA
splicing factors that can also influence the selection
of alternative 5′ splice sites in a concentration-dependent
manner. Their activity in alternative splicing in vitro
is antagonized by members of the hnRNP A/B family of proteins.
The opposite effects of members of these two families of
antagonistic splicing factors in vitro and upon overexpression
in vivo suggest that changes in their relative levels may
be a natural mechanism for the regulation of alternative
splicing in vivo. One prediction of this model is that
the ratios of these antagonists should vary in different
cell types and in other situations in which cellular or
viral transcripts are differentially spliced. We raised
monoclonal antibodies specific for SF2/ASF and used them
to measure the abundance of SF2/ASF protein and its isoforms,
its phosphorylation state in vivo and during splicing in
vitro, and its association with the spliceosome. SF2/ASF
exists predominantly or exclusively in a highly phosphorylated
state in vivo in all cell types examined, and unphosphorylated
protein was not detectable. Unphosphorylated recombinant
SF2/ASF becomes rapidly phosphorylated under splicing conditions
in HeLa cell extracts and associates stably with one or
more exons of β-globin pre-mRNA. This interaction appears
to persist through the splicing reaction and SF2/ASF remains
bound to spliced mRNA. We compared the distribution of
SF2/ASF to that of its antagonist, hnRNP A1, in different
rat tissues and in immortal and transformed cell lines.
We found that the protein levels of these antagonistic
splicing factors vary naturally over a very wide range,
supporting the notion that changes in the ratio of these
proteins can affect alternative splicing of a variety of
pre-mRNAs in vivo.