Post-transcriptional Regulation of Gene 
Expression, Rihonucleoprotein Complexes, 
and Nuclear Structures 
Gideon Dreyfttss, Ph.D. — Investigator 
Dr. Dreyfuss is also Professor of Biochemistry and Biophysics at the University of Pennsylvania School of 
Medicine. He received his Ph.D. degree in biological chemistry from Harvard University and was a Helen 
Hay Whitney postdoctoral fellow at the Massachusetts Institute of Technology. Prior to his present ap- 
pointment he was Professor and Established Investigator of the American Heart Association at North- 
western University. 
MESSENGER RNAs (mRNAs), the functional 
translatable intermediates of gene expres- 
sion, are formed in the nuclei of eukaryotic cells 
by extensive and tightly regulated post-transcrip- 
tional processing of primary RNA polymerase II 
transcripts. These transcripts are termed hetero- 
geneous nuclear RNAs (hnRNAs), which de- 
scribes their variable size and cellular localiza- 
tion. It is possible that only a subset of hnRNAs 
are actually precursors of mRNA and that the rest 
turn over in the nucleus. 
The collective term for the proteins that bind 
hnRNAs, and that are not stable components of 
other classes of rihonucleoprotein (RNP) com- 
plexes, is hnRNP proteins. They are significant in 
that they are bound to hnRNAs and thus influence 
their structure, fate, and processing into mRNAs. 
The hnRNP proteins are also as abundant in grow- 
ing vertebrate cells as histones, and their com- 
plexes with hnRNA are also of interest as major 
nuclear structures. 
Once formed, the mRNAs are transported to the 
cytoplasm via nuclear pores — a little-known pro- 
cess that appears to be one of the most important 
regulatory steps in the post-transcriptional path- 
way of gene expression. In the cytoplasm, mRNP 
proteins are likely to be involved in regulating 
the translation, stability, and localization of 
mRNAs. Our goal is to understand, in molecular 
detail and cellular architecture, how the post- 
transcriptional portion of the pathway of gene 
expression operates in the cell. To do so we in- 
vestigate the structure, function, and localization 
of the hnRNP and mRNP proteins and the RNP 
complexes. 
We have identified the hnRNP and mRNP pro- 
teins by photochemical RNA-protein crosslinking 
in intact cells and have produced monoclonal an- 
tibodies to many of them. The antibodies were 
used to develop an immunopurification proce- 
dure for hnRNA-hnRNP complexes from verte- 
brate cells and to begin characterizing the pro- 
teins. Human cells have provided the most 
detailed picture of the protein composition of 
these complexes. Considerable information is 
also becoming available for invertebrates, partic- 
ularly Drosophila. Immunopurified complexes 
contain large RNA of up to 10,000 nucleotides 
and at least 20 major proteins, designated A-U, in 
the range of 34-120 kDa. There are also many 
lower-abundance hnRNP proteins, and these ap- 
pear to bind only to specific subsets of hnRNAs. 
Nuclease digestion experiments indicate that the 
hnRNP proteins are not simply dispersed on the 
hnRNAs, but rather are organized into interacting 
units. 
The molecular cloning and sequencing of 
cDNAs for several RNP proteins led to the discov- 
ery of a conserved RNA-binding domain (RBD) 
and a rihonucleoprotein consensus sequence 
(RNP-CS). The RNP-CS has subsequently been 
found in RNP proteins from many sources. This 
octapeptide sequence, Lys/Arg-Gly-Phe/Tyr-Gly/ 
Ala-Phe-Val-X-Phe/Tyr, is the most highly con- 
served segment in a generally conserved region of 
about 90-100 amino acids present at least once 
in many RNA-binding proteins of the nucleus and 
cytoplasm in organisms as evolutionarily diver- 
gent as yeast, plants, and humans. This region, 
because of its general conservation as a unit, was 
considered to be an RNA-binding domain, and the 
RNP-CS was suggested to be an important ele- 
ment in the RNA-binding activity of the RBD. 
These predictions have turned out to be correct, 
and the predictive value of consensus sequences 
for the identification of proteins as having RNA- 
binding activities has proved very useful. 
We have produced the 94-amino acid RBD of 
the hnRNP CI protein and purified it. The three- 
dimensional structure of this domain and of its 
complex with RNA oligonucleotide substrate are 
being determined. 
Experiments on mitotic cells unexpectedly 
provided important insights into the assembly 
and general nature of hnRNP complexes. In mito- 
sis, as the nuclear envelope breaks down, hnRNP 
proteins disperse throughout the cell, but they 
remain associated in complexes with RNA. After 
mitosis, once the nuclear envelope reforms, 
preexisting hnRNP proteins return to the nu- 
cleus. Double-label immunofluorescence micros- 
copy with monoclonal antibodies to various 
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