Molecular Analysis of Muscle Development and Function 
2l recently identified MADS gene family, which 
includes yeast transcription factors, plant homeo- 
tic genes involved in floM^er morphogenesis, and 
the serum response factor. 
MEF2 mRNAs accumulate preferentially in skel- 
etal muscle, heart, and brain, but post-transcrip- 
tional mechanisms must also contribute to the 
, MEF2 tissue specificity. Remarkably, cardiac and 
smooth as well as skeletal muscle contain saturat- 
ing levels of MEF2 trans-activating factors that are 
absent in nonmuscle cells. We have shown that 
MEF2 is induced in skeletal muscle cells by the 
MyoD gene family, but that this factor, by itself, is 
insufficient to produce the whole muscle pheno- 
type. Therefore, although MEF2 is induced in 
skeletal muscle by bHLH proteins, other lineage- 
determining pathways must lead to MEF2 expres- 
sion in nonskeletal muscle tissues. 
In this context, the finding that both muscle 
and brain have high-level expression of the same 
isoforms strongly suggests that MEF2 also has an 
important role in neuronal gene regulation. 
Whether bHLH or other factors induce MEF2 
genes in cardiac and smooth muscle, as well as 
nerve, the regulatory sequences of these genes 
will serve as powerful tools for the identification 
of the lineage-determining pathways in these cell 
types. 
Regulation of Protein Diversity 
by Alternative Splicing 
One of the fundamental aspects of pre-mRNA 
splicing that is particularly important for the gen- 
eration of different proteins from a single gene is 
the mechanism by which splice sites are identi- 
fied. We have previously suggested a scanning 
model for the location of the 3' splice site of 
mammalian introns. We proposed that this site is 
located by a scanning mechanism searching for 
the first AG downstream of the branch point/ 
polypyrimidine tract. During the past year we 
have further confirmed and extended this model. 
Recent experiments show that scanning for the 
3' splice site starts at the branch point, not the 
pyrimidine tract, and proceeds until an AG is rec- 
ognized. Failure to recognize the most proximal 
AG can arise from extreme proximity to the 
branch point, or the AG can be sequestered 
within a hairpin loop. Once the AG has been en- 
countered, scanning stops, but the spliceosome 
can still see a stretch of about 1 2-35 nucleotides 
downstream. The most competitive AG within 
this scanning window is then selected as the 3' 
splice site. The strongest determinant of compe- 
titiveness remains the proximity to the branch 
point. Thus, in many respects, the scanning 
model for the 3' splice site closely resembles the 
model for translation initiation, both in its sim- 
plest formulation and in the predictable excep- 
tions to the general rule. 
To analyze the mechanisms involved in alterna- 
tive splicing, we have continued to focus on the 
a-tropomyosin (TM) gene. This gene generates a 
minimum of 1 0 different isoforms that are tissue 
specific and developmentally regulated. We have 
concentrated on the mutually exclusive exons 2 
and 3 to elucidate the elements involved in this 
type of regulation. 
In the past we identified the mechanisms in- 
volved in the production of the default pattern, 
which results in the exclusion of exon 2 and in- 
clusion of exon 3 in all cell types but smooth 
muscle cells. During the past year, we have con- 
centrated on the production of the regulated pat- 
tern in smooth muscle cells that results in the 
inclusion of exon 2 and exclusion of exon 3- This 
pattern is the result of negative regulation by inhi- 
bition of the default pattern and is attributable to 
two well-defined sequences located 5' and 3' 
from exon 3- These sequences are the binding 
site for protein factors present in smooth muscle 
but not in HeLa cells. Experiments are in progress 
to clone this factor. 
The search for splicing factors that interact 
with the polypyrimidine tract has resulted in the 
cloning and characterization of a novel essential 
splicing factor. This is a protein of about 1 00 kDa 
that is closely associated with the polypyrimidine 
tract-binding protein previously described by 
our laboratory and others. Experiments are in pro- 
gress to determine the particular stage of spliceo- 
some assembly in which this factor is involved. 
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