ing of cDNAs for the hnRNPA2, Bl, CI, C2, I, K, L, 
and U proteins. Recently, cDNAs of the hnRNP F, 
G, H, and M proteins have been isolated. The hn- 
RNP complexes of D. melanogaster have also 
been isolated, and their major proteins have been 
sequenced. 
Structure of hnRNPs 
Sequence analysis, mutagenesis, and binding stud- 
ies of hnRNPs delineated several motifs for RNA 
binding and protein-protein interaction. Most of the 
hnRNPs (as well as many mRNA-, pre-rRJVA- and 
snRNA-binding proteins) belong to a large family of 
RNA-binding proteins that contain one or several 
highly conserved 90- to 100-amino acid RNP mo- 
tifs that define an RNA-binding domain (RBD). Dr. 
Dreyfuss and his colleagues (in collaboration v^^ith 
Drs. Michael Wittekind, Robert A. Beckman, and Lu- 
ciano Mueller of Bristol-Myers Squibb) have deter- 
mined the structure of the 93-amino acid RBD of 
the pre-mRNA-binding hnRNP C protein in solution 
and mapped candidate amino acids involved in RNA 
binding by multidimensional nuclear magnetic 
resonance (NMR). This RBD has a compact folded 
structure 0a0-fiafi) comprising a four-stranded anti- 
parallel /3 sheet, two a helices, and relatively un- 
structured amino- and carboxyl-terminal regions. 
The RNPl and RNP2 consensus sequences — which 
are the most highly conserved peptides of this RNP 
family — are juxtaposed on the adjacent central /3 
strands (|83 and i8l) of the /? sheet and exposed on 
the surface of the domain. In vitro random- 
sequence selection methods were used to identify 
specific RNA ligands for this RBD. These experi- 
ments demonstrated that the hnRNP C proteins bind 
to oligo-uridine-containing RNA. 
The interaction of the hnRNP C protein RBD with 
r(U)8 was studied in NMR experiments. These stud- 
ies revealed that residues in the jS-sheet region and 
in the amino- and carboxyl-terminal regions of the 
RBD are significantly affected by the formation of 
the hnRNP C RBD:r(U)8 complex. In contrast, the 
residues of the well-conserved a helices, with one 
exception, were not affected. Mutagenesis is being 
used to identify independently residues within the 
RBD that are critical for its interactions with RNA. 
These analyses indicate that a large number of amino 
acids on the /? sheet and the contiguous amino- and 
carboxyl-terminal regions of the RBD provide an ex- 
posed "platform" for extensive interactions with 
the RNA. RNA bound to this platform should be ac- 
cessible to other (e.g., splicing) factors, rather than 
buried in a binding pocket. 
Localization and Transport 
of pre-mRNA-binding Proteins 
Immunofluorescence microscopy with most of 
the hnRNP antibodies shows general nucleoplasmic 
localization for these proteins with little or no stain- 
ing in the nucleoli and in the cytoplasm. Much of 
this signal results from staining of nascent tran- 
scripts; experiments on Drosophila polytene chro- 
mosomes allow observation of the association of 
individual hnRNPs and snRNPs with specific pre- 
mRNAs. The nuclear staining was interpreted to indi- 
cate that hnRNPs are restricted to the nucleus of 
interphase cells, with the conclusion that the func- 
tions of hnRNPs concern strictly nuclear processes. 
However, Dr. Dreyfuss's laboratory recently 
found that this is not always the case and that some 
of the hnRNPs (such as those of the A and B groups) 
shuttle between the nucleus and the cytoplasm. In 
contrast, other hnRNPs (such as the C and U pro- 
teins) appear to be confined to the nucleus. This 
suggests that the shuttling hnRNPs may also have 
functions in the cytoplasm, that the hnRNP com- 
plexes are dynamic, and that a role for these pro- 
teins in nucleocytoplasmic transport of RNA must 
be considered. All of the hnRNPs must be imported 
into the nucleus, and some of them, the shuttling 
proteins, must also be exported to the cytoplasm. A 
role for polymerase II transcription in the localiza- 
tion of these proteins has also receiitly emerged. 
The signals in the hnRNPs that mediate their trans- 
port and localization are being investigated. 
Dr. Dreyfuss is also Professor of Biochemistry 
and Biophysics at the University of Pennsylvania 
School of Medicine. 
Articles 
Bennett, M., Piiiol-Roma, S., Staknis, D., Dreyfuss, 
G., and Reed, R. 1992. Differential binding of het- 
erogeneous nuclear ribonucleoproteins to mRNA 
precursors prior to spliceosome assembly in vi- 
tro. Mol Cell Biol 12:3165-3175. 
Ghetti, A., Pinol-Roma, S., Michael, W.M., Mor- 
andi, C., and Dreyfuss, G. 1992. hnRNP I, the 
polypyrimidine tract-binding protein: distinct 
nuclear localization and association with hnRNAs. 
Nucleic Acids Res 14:3671-3678. 
Kiledjian, M., and Dreyfuss, G. 1992. Primary 
structure and binding activity of the hnRNP U 
protein: binding RNA through RGG box. EMBO J 
11:2655-2664. 
Matunis, E.L., Matunis, M.J., and Dreyfuss, G. 
CELL BIOLOGY AND REGULATION 47 
