number of genes that contained sequences similar 
to the E2F recognition site. These include genes 
such as the c-myb proto-oncogene, the N-myc 
proto-oncogene, the DHFR gene, and the EGF re- 
ceptor gene. These genes are commonly regulated 
during cell proliferation, suggesting a role for E2F 
in this process. Indeed, serum-stimulated expres- 
sion of the c-myc promoter in 3T3 cells is depen- 
dent on the E2F sites. Furthermore, the DNA-bind- 
ing activity of E2F is increased fourfold upon serum 
stimulation, and this increase is independent of 
new protein synthesis, as is the stimulation of 
c-myc transcription upon serum stimulation. 
II. Control of RNA Processing. 
In addition to the control of transcription initia- 
tion, it is now clear that post-transcriptional con- 
trol of gene expression can be an important mech- 
anism of gene regulation. Often a transcription 
unit encodes not one mRNA but several mRNAs. 
Through alternative processing of the primary tran- 
script, one particular mRNA, and thus one particu- 
lar protein, is selected and produced. In several 
cases this alternative processing has been shown to 
be a regulated event subject to change, depending 
on the circumstances in the cell. Alternative pro- 
cessing can involve both the selective splicing of 
exons and the selection of one of several poly(A) 
addition sites, thus generating the mRNA 3' termi- 
nus. Possibly the best example of regulation of 
poly(A) site utilization is within the differentiating B 
lymphocyte, where there is a dramatic change in 
the nature of immunoglobulin heavy-chain poly- 
peptides that are synthesized. The RNAs that direct 
the synthesis of the two forms of heavy-chain pro- 
teins are encoded in the same transcription unit 
and are produced by the differential cleavage of the 
primary transcript at two different poly(A) sites. A 
definition of the events and factors involved in 
poly(A) site formation using transfection assays and 
in vitro systems has been a major goal of the labo- 
ratory. 
Recently efforts have been directed at the analy- 
PUBLICATIONS 
sis of cell-free systems that carry out poly(A) site 
processing in vitro to isolate factors involved in this 
reaction and define their role in the processing 
event. Four HeLa cell nuclear factors that are 
required for specific pre-mRNA cleavage and poly- 
adenylation have now been extensively purified. 
Two factors, PFl and PF2, are required for specific 
polyadenylation of the cleaved RNA. PFl is a 
poly(A) polymerase, and PF2 is a factor that confers 
specificity to the reaction by allowing recognition 
of the AAUAAA sequence, an element that is essen- 
tial for poly(A) site utilization. Both of these factors, 
along with two additional factors, CFl and CF2, 
are required for the endonucleolytic cleavage of 
the pre-mRNA. The ability of each of these factors 
to form specific complexes with the pre-mRNA 
has been assayed using native gel electrophoresis. 
Two distinct complexes were detected. PF2, the 
specificity factor, forms an initial complex with 
the pre-mRNA dependent on the AAUAAA sequence 
element but independent of specific downstream 
sequences. Formation of the PF2 RNA complex 
permits the subsequent interaction of CFl and 
the formation of a second larger complex. CFl 
binding requires the downstream sequence ele- 
ment in addition to PF2 binding. Whereas the PF2 
RNA complex is unstable and rapidly dissociates, 
the ternary complex formed by a CF1/PF2 and the 
RNA is stable. Thus the interaction of CFl depen- 
dent on the downstream sequence element can be 
viewed as a commitment of the poly(A) site for 
processing. Upon the addition of the poly(A) poly- 
merase (PFl) and the CF2 factor, the pre-mRNA is 
specifically cleaved at the poly(A) site and then 
polyadenylated. With these factors in hand and 
distinct assays for each factor available, the possi- 
bility exists of assaying a regulated system such as 
the differentiating lymphocyte to define which fac- 
tor is the rate-limiting component in poly(A) site 
utilization. 
Dr. Nevins is also Professor of Microbiology and 
Immunology at the Duke University Medical 
Center. 
Books and Chapters of Books 
Guise, J.W, Galli, G., Nevins, J.R., and Tucker, RW 1988. Developmental regulation of secreted and mem- 
brane forms of immunoglobulin |x chain. In Immunoglobulin Genes (Honjo, T, Ed.). New York: Academic, 
pp 275-301. 
Continued 
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