POST-TRANSCRIPnONAL CONTROLS OF EUKARYOTIC GENE EXPRESSION 
Stephen A. Liebhaber, M.D., Associate Investigator 
Dr. Liebhaber's laboratory studies the structure 
and function of eukaryotic genes, specifically the re- 
lationship of mRNA structure to its processing and 
function. These studies are carried out in two well- 
characterized model systems: the human a-globin 
gene family and the human growth hormone gene 
family. 
I. Structural Basis for Splice-Site Selection. 
Primary structural signals necessary for accurate 
splicing of RNA polymerase II transcripts are now 
defined in detail. It is clear, however, that these 
minimal signals, while necessary for splicing, are 
not sufficient and that additional signals— primary 
sequence as well as higher order structures— also 
appear to have a role in splice-site selection. The 
normal human pituitary growth hormone gene 
(hGH-N) consists of five exons. Two splice acceptor 
sites are used in intron 2, a major acceptor (B), 
used 90% of the time, and an alternative splice ac- 
ceptor (B') located 45 bases within exon 3 (B'), 
used —10% of the time. The alternatively spliced 
mRNA encodes an hGH protein with a 15 -amino 
acid internal deletion that may have physiologic 
functions distinct from the normal 22 kDa hGH. In 
contrast to this splicing pattern, the highly similar 
placental hGH-variant gene (hGH-V), fails to utilize 
the B' acceptor site, despite an identical sequence 
surrounding both B and B' splice acceptor sites. 
This laboratory is exploring the structural basis for 
this difference in the splicing patterns of these two 
highly related genes. 
In an initial study, both hGH-N and hGH-V genes 
have been expressed in a variety of cell lines. Both 
genes maintain their respective splicing patterns in 
these lines, demonstrating that the splicing pat- 
terns are neither developmentally regulated nor 
tissue specific. Exon 3 and surrounding intron 
sequences were exchanged between these two 
genes, in order to map the determinants of the 
exon 3 splicing pattern. This exchange resulted in a 
parallel exchange in the splicing patterns. This 
suggested that exon 3 and/or adjacent intron 2 
sequences contain essential determinants of splice- 
site selection. 
To define these determinants more accurately. Dr. 
Liebhaber and his colleagues have introduced site- 
specific mutations within intron 2 and exon 3. Re- 
sults demonstrate that the alternative splice is abso- 
lutely dependent on the presence of a single, spe- 
cific adenosine (A) located 22 bases upstream of 
the B' acceptor site. This A is present in hGH-N but 
not in hGH-V Whether this base is the lariat branch 
A of the alternative splice acceptor is now being in- 
vestigated. Additional mutations suggest that other 
sequences in the environment of the B and B' 
splice sites can significantly alter the probability 
with which the alternative splice site is used. Stud- 
ies now under way are focusing on the position and 
mode of action of these additional signals. 
II. Relationship of mRNA Structure to Translational 
Efficiency. 
Each mRNA species may have its own level of 
translational efficiency. Although the primary struc- 
tural signals important in accurate translation initi- 
ation and termination have been known for some 
time, the structures that determine translational ef- 
ficiency remain poorly defined. Dr. Liebhaber's lab- 
oratory is studying these signals in a well-character- 
ized model system. The a- and ^globin genes 
encode the two protein subunits of hemoglobin. 
Equal quantities of a- and P-globin (a2^2) are syn- 
thesized in the developing erythrocyte, despite a 
severalfold excess of a-globin mRNA. This balanced 
protein synthesis reflects a higher translational effi- 
ciency of (3-globin mRNA. Initial experiments have 
demonstrated that P-globin mRNA is present on 
heavier polysomes than a-globin mRNA, despite 
their approximately equal sizes (145 and 141 co- 
dons, respectively). In vitro translation studies 
demonstrate that this difference in polysome load- 
ing reflects their relative abilities to load ribosomes 
during steady-state translation, as opposed to initial 
monosome assembly. Measurement of ribosome 
binding when the mRNAs are hybridized to cDNA 
fragments covering specific regions (hybrid arrest of 
translation initiation) demonstrated that the ribo- 
some assembly site of a-globin mRNA requires 
more unimpeded space 3' to the initiation codon 
than does ^-globin mRNA to assemble an 80 S ribo- 
some. This suggests a difference in the structure of 
the two respective ribosome assembly sites, which 
could impart a relative delay in ribosome loading of 
a-globin mRNA during steady-state translation. The 
5'-nontranslated and 5' proximal coding regions of 
these two mRNAs have been exchanged and chime- 
ric a/p-globin mRNAs have been synthesized in 
order to map the regions responsible for this differ- 
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