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The goal of functional genomics is to determine the function
of each protein encoded by an organism. Typically, this is done
by inactivating individual genes and, subsequently, analyzing
the phenotype of the modified organisms. In higher eukaryotes,
where a tremendous amount of alternative splicing occurs, such
approaches are not feasible because they have the potential
to simultaneously affect multiple proteins that could have quite
distinct and important functions. Thus, it is necessary to develop
techniques that inactivate only a subset of proteins synthesized
from genes encoding alternatively spliced mRNAs. Here we
demonstrate that RNA interference (RNAi) can be used to selectively
degrade specific alternatively spliced mRNA isoforms in cultured
Drosophila cells. This is achieved by treating the
cells with double-stranded RNA corresponding to an alternatively
spliced exon. This technique may prove to be a powerful tool
to assess the function of proteins synthesized from alternatively
spliced mRNAs. In addition, these results have implications
regarding the mechanism of RNAi in Drosophila.
Splicing enhancers are RNA sequence elements that promote
the splicing of nearby introns. The mechanism by which
these elements act is still unclear. Some experiments support
a model in which serine-arginine (SR)-rich proteins function
as splicing activators by binding to enhancers and recruiting
the splicing factor U2AF to an adjacent weak 3′ splice
site. In this model, recruitment requires interactions
between the SR proteins and the 35-kDa subunit of U2AF
(U2AF35). However, more recent experiments have
not supported the U2AF recruitment model. Here we provide
additional evidence for the recruitment model. First, we
confirm that base substitutions that convert weak 3′
splice sites to a consensus sequence, and therefore increase
U2AF binding, relieve the requirement for a splicing activator.
Second, we confirm that splicing activators are required
for the formation of early spliceosomal complexes on substrates
containing weak 3′ splice sites. Most importantly,
we find that splicing activators promote the binding of
both U2AF65 and U2AF35 to weak 3′
splice sites under splicing conditions. Finally, we show
that U2AF35 is required for maximum levels of
activator-dependent splicing. We conclude that a critical
function of splicing activators is to recruit U2AF to the
weak 3′ splice sites of enhancer-dependent introns,
and that efficient enhancer-dependent splicing requires
Members of the serine/arginine-rich (SR) protein
family have multiple functions in the pre-mRNA splicing
reaction. In addition to being required for the removal
of constitutively spliced introns, SR proteins can function
to regulate alternative splicing both in vitro and in vivo
(Ge & Manley, 1990; Krainer et al., 1990a; Fu et al.,
1992; Zahler et al., 1993a; Caceres et al., 1994; Wang
& Manley, 1995). In the cell, SR proteins migrate from
speckles—subnuclear domains that may function as
storage sites for certain splicing factors—to sites
of active transcription (Misteli et al., 1997; Misteli
& Spector, 1999) and some SR proteins have been found
to shuttle in and out of the nucleus (Caceres et al., 1998).
The subcellular localization of SR proteins can be modulated
by phosphorylation (Misteli & Spector, 1998; Misteli
et al., 1998) and this undoubtedly underlies some regulated
splicing events. However, once in the nucleus and localized
to the nascent pre-mRNA, exactly how SR proteins engage
the general splicing machinery to recognize specific splice
sites is unclear and is an area of intense investigation.
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