Gene Regulation in Animal Cells 
is a critical event in the biogenesis of most mRNA 
molecules. Moreover, since many transcription 
units encode multiple mRNAs that utilize alterna- 
tive poly(A) sites, there is a potential regulatory 
role for polyadenylation. Indeed, analysis of the 
formation of the immunoglobulin heavy-chain 
transcripts during B cell differentiation suggests 
-that alternative poly(A) site choice is a contribut- 
ing factor in the switch from production of the Ig 
mRNA that encodes the membrane-bound form of 
the protein to the mRNA encoding the secreted 
form of the protein. Thus an understanding of the 
mechanism of poly(A) site utilization, including 
the sequences directing the processing event and 
the factors involved in processing, is an impor- 
tant focus of our work. 
We have recently used a biochemical approach 
to investigate the molecular mechanism of poly- 
adenylation. Factors have been purified from 
HeLa cell nuclear extracts that can reconstitute 
accurate and sequence-specific poly(A) site pro- 
cessing in vitro. Two of these factors interact spe- 
cifically with the pre-mRNA, and an analysis of 
this interaction suggests a pathway of assembly of 
a functional poly (A) site-processing complex. 
The interaction of one factor with the con- 
served AAUAAA element is specific, but the RNA- 
protein complex that forms is very unstable and 
rapidly dissociates. The interaction of a second 
factor with the RNA requires a distinct sequence 
element but also requires the prior interaction of 
the first factor with the AAUAAA element. Most 
importantly, the resulting ternary complex is 
stable and does not readily dissociate. 
These observations suggest a pathway of assem- 
bly of poly(A) site factors that involves an initial 
recognition of the AAUAAA element followed by a 
commitment step in which the second factor sta- 
bilizes the overall complex. Since the relative sta- 
bility of this complex reflects the relative effi- 
ciency of processing, it appears that these 
interactions may be important regulatory targets 
for the control of polyadenylation. 
This work on the mechanisms of polyadenyla- 
tion is supported by a grant from the National 
Institutes of Health. 
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