Post-transcriptional Regulation of Gene Expression, RNA-Protein Complexes, 
and Nuclear Structures 
sufficient for RNA binding. This RBD was pro- 
duced in bacteria and purified to homogeneity in 
active form. Nuclear magnetic resonance (NMR) 
methods (in collaboration with Luciano Mueller 
and Michael Wittekind, Bristol-Myers Squibb), 
including '^C- and ^^N-edited three-dimensional 
NMR, were used to determine the structure of the 
RBD in solution. The compact folded structure 
exhibits a four-stranded antiparallel |8-sheet and 
two well defined a-helices. The structure of this 
RBD complexed with an RNA substrate is being 
determined. 
Experiments on mitotic cells unexpectedly 
provided important insights into the assembly 
and general nature of hnRNP complexes and into 
the transport of proteins to the nucleus. In mito- 
sis, as the nuclear envelope breaks down, hnRNPs 
disperse throughout the cell but remain asso- 
ciated in complexes with RNA. After mitosis, 
once the nuclear envelope re-forms, preexisting 
hnRNPs return to the nucleus. We observed, how- 
ever, using double-label immunofluorescence 
microscopy with monoclonal antibodies to 
various hnRNPs on postmitotic cells, that at the 
end of mitosis the hnRNP complexes dissociate in 
the cytoplasm and the different proteins are trans- 
ported to the nucleus separately. Some, includ- 
ing CI, C2, and U, like snRNPs and lamins, are 
transported immediately (early group), while 
others, including Al, A2, Bl, B2, E, G, H, and L, 
are transported into the nucleus later (late 
group). Thus, immediately following reassembly 
of the nuclear envelope at the end of mitosis, 
pairs of cells are detected in which some hnRNPs 
are in the nucleus, and others are in the cyto- 
plasm. These observations show that hnRNP com- 
plexes are dynamic structures, in that hnRNPs 
can dissociate from the complexes and return to 
the nucleus separately. 
Surprisingly, the transport of the late group re- 
quires transcription by RNA polymerase II: inhibi- 
tors of this polymerase cause the late proteins to 
remain in the cytoplasm. Thus there are two path- 
ways for nuclear transport localization of pro- 
teins: a transcription-independent pathway and a 
novel transcription-dependent pathway. The dif- 
ferent hnRNPs utilize one of the two pathways, 
and both pathways operate throughout the cell 
cycle. The signals in the proteins that spec- 
ify the pathway to be used, the mechanism of 
transcription-dependent transport localization, 
and the relevance to mRNA transport are being 
investigated. 
Immunofluorescence microscopy indicated 
that the hnRNP proteins A, B, C, E, I, K, L, M, and 
U are nucleoplasmic — that is, localized to the 
nucleus but excluded from nucleoli. In contrast, 
the mRNPs that have been characterized so far [in 
particular the poly (A) -binding protein] are con- 
fined to the cytoplasm. It was therefore con- 
cluded that the mRNA must exchange most if not 
all of the proteins with which it is associated in 
the nucleus as it is transported to the cytoplasm. 
The dissociation of these proteins from the mRNA 
and the subsequent binding of mRNPs must be an 
important aspect of nuclear-cytoplasmic trans- 
port of mRNA. 
Recently we found that several of the abundant 
hnRNPs, including Al, are not confined to the 
nucleus but rather shuttle continuously between 
the nucleus and the cytoplasm. Thus hnRNPs may 
have cytoplasmic functions in addition to their 
nuclear roles in the processing of pre-mRNA to 
mRNA. Furthermore, Al is bound to mRNA in the 
cytoplasm and its return to the nucleus requires 
RNA polymerase II transcription. It is therefore 
possible that the novel cytoplasmic ribonucleo- 
protein complex of mRNA with hnRNPs is the 
substrate of RNA nuclear-cytoplasmic transport. 
Thus it is likely that hnRNPs play an active role 
in mRNA export and that the mRNA is transported 
to the cytoplasm as a result of its association with 
the shuttling hnRNPs. Understanding the traffick- 
ing of hnRNPs in the cell and the mechanisms 
that regulate the assembly and disassembly of 
RNPs with hnRNAs and mRNAs should be impor- 
tant for elucidating the post-transcriptional path- 
way of gene expression, including the nuclear 
transport process of mRNA. 
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