Download From the Nucleus Toward the Cell Periphery: a Guided

From the Nucleus Toward the Cell Periphery:
a Guided Tour for mRNAs
Brigitte M. Jockusch,1 Stefan Hüttelmaier,1,2 and Susanne Illenberger1
1
Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany; and 2Albert Einstein College of Medicine, New
York, New York 10461
T
issue generation during embryogenesis and wound healing
involves cell proliferation as well as cellular differentiation. The latter comprises a variety of highly complex
processes, culminating in structural and functional polarity of
the tissue-forming cells. The most important steps are differential gene activation and transcription, RNA splicing and maturation, and, finally, site-specific translation of tissue-specific
proteins. Recent evidence suggests that these steps may be
coupled, which would increase the efficiency of the assembly
of peripheral protein complexes. However, temporal as well
as spatial coupling requires mediators. For example, how are
nuclear processes, like RNA generation and processing, wired
to steps occurring at the cell periphery, like the assembly of
plasma membrane-associated protein complexes by site-specific protein synthesis? The discovery of specific “dual-compartment” proteins that participate in nuclear as well as in
peripheral processes may provide an answer to this question.
Such proteins could act as tour guides for mRNAs traveling
from one compartment to the other. The guiding process itself,
however, is complex and far from understood today.
Transport vehicles, traffic, and cargo delivery
Site-specific localization of mRNA in cells, as a result of
either directed transport, concentration by a specific anchoring mechanism, or region-specific protection from degradation, has been observed in a large number of cases, in particular in oocytes and early embryos of vertebrates and insects,
but it has also been observed in yeast, where these mechanisms result in targeting of proteins engaged in cell and tissue
polarization or the determination of cell fate (1, 15). To better
understand the mechanism and the biological significance of
the directed transport of mRNAs in all of these systems, one
would need to know the exact composition of their cytoplasmic cages (“locasomes”; Ref. 3), the relation between specific
cargo mRNAs and their locasomes, the nature of the tracks,
and the linker as well as the motor proteins that might drive
the polarized transport. Regarding the tracks, there is good
evidence that mRNA movement utilizes the cytoskeleton (8).
Of the three major cytoskeletal filament systems expressed in
animal cells [intermediate filaments, microtubules, and micro0886-1714/03 5.00 © 2003 Int. Union Physiol. Sci./Am. Physiol. Soc.
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RNA processing, directed transport along cytoskeletal tracks, and site-specific translation of mRNA
at the cell periphery are considered discrete steps in the generation of microfilament-membrane
adhesion complexes. A recently identified member of the heterogeneous nuclear ribonucleoprotein family, raver1, may couple these steps and contribute to the assembly and maintenance of these structures.
filaments] two, namely microtubules and microfilaments, participate in the directed transport of mRNAs. The mRNA-containing protein complexes are tied to cytoskeletal fibers, transported along them, and also translated when still tethered to
these structures (e.g., Ref. 4). Apparently, the choice of the
tracks is not so much dependent on the nature of the mRNA
transported but on the cell type, and a switch from one to the
other cytoskeletal track system also seems possible (1, 15).
Accordingly, both types of cytoskeletal motor enzymes, i.e.,
kinesins and myosins, may be involved. Within this field, the
transport of cytoskeletal mRNAs raises a particularly interesting question: do the cytoskeletal elements serve as tracks to
move their own mRNAs and thus contribute to the directed
translation of their own components? This might be an efficient mechanism in the assembly of peripheral structures,
such as microfilament-plasma membrane attachment sites in
adhesive junctions that play a crucial role in tissue generation.
Having reached their destination and at the end of their
motor-assisted journey, at least some components of the locasomes must be anchored and, finally, their cargo mRNA
should be translated. In this context, the identification of the
elongation factor EF1 as an actin filament- and -actin
mRNA-binding protein is noteworthy, because it may couple
anchoring of actin mRNA to the cytoskeletal fibers with translation and thus allow for cotranslational assembly of
cytoskeletal structures in distal parts of the cell (12).
b-Actin is the actin isoform associated with dynamics and
motility of the cell periphery, and -actin mRNA was seen
concentrated at the leading edge of locomoting fibroblasts
(10) and at the tips of neurites (2). The interaction between this
mRNA and proteins engaged in its transport through the cytoplasm has been studied in detail. Within the 3'-untranslated
region, a 54-nt motif, the “zip code,” was found to interact
with several zip code-binding proteins, and this interaction
seems essential for transport, localization, and translation of actin mRNA in fibroblasts and neurons (6). One of these proteins, ZBP2, is an avian ortholog of the human protein KHtype splicing regulatory protein (KSRP), is a member of the
heterogeneous nuclear ribonucleoprotein (hnRNP) family (see
below), and is predominantly, but not exclusively, located in
the nucleus (6). Another interesting case of complexes
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The common view on how PTB/hnRNPI and related proteins
can achieve so many functions is of course that they associate
with different ligand proteins, contributing to discrete multiprotein complexes that then participate in only a few or even
one of these functions. Indeed, hnRNPs form homo- and
heterooligomeric complexes with other members of the family, but a complete catalog of their ligands is still missing. It is
also not known whether, in general, shuttling hnRNPs are
packaged and transported along with the mRNAs, and, if so,
whether these are the same hnRNPs that already participated
in the generation of the particular mRNA that is cotransported.
Raver1, an hnRNP protein with unique properties
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The recent discovery of raver1 (7), another hnRNPI protein,
FIGURE 1. Raver1 colocalizes with pyrimidine tract-binding protein
(PTB)/heterogeneous nuclear ribonucleoprotein I (hnRNPI) in perinucleolar
bodies of myoblasts. Double fluorescence images obtained with antibodies
against PTB/hnRNPI (left) and raver1 (middle) show that, in undifferentiated
C2C12 myoblasts, both proteins reside within the nuclear matrix but not
within nucleoli (black spots). Both are concentrated in the perinucleolar bodies (arrowheads), and their exact colocalization is demonstrated in the overlay images (right). Bars = 10 m. (Reprinted with permission from Ref. 7).
between mRNAs and peripheral proteins is represented by
recent data demonstrating that PABP1, a protein that binds to
and stabilizes the poly(A) tail of a variety of mRNAs, forms
high-affinity complexes with paxillin, a microfilament adaptor
protein involved in the regulation of cell motility (20).
The hnRNP family: multitalented tour guides
These findings indicate that the multiple steps from nuclear
RNA maturation to peripheral translation may be guided by
proteins that are multifunctional and act as chaperones for
mRNAs on their journey from the nucleus to the periphery. In
the search for candidates mediating between mRNAs and protein components of transport particles, a number of RNAbinding proteins of the hnRNP family (5) have gained much
attention. The hnRNPs display protein-specific subsets of conserved RNA-binding domains, including RNA-recognition
motifs (RRMs), heterogeneous nuclear ribonucleotide protein
K (hnRNPK) homology domains (KH domains), and glycinearginine-rich domains that are involved in mediating specific
protein-RNA and protein-protein interactions. These proteins
bind to premature and mature mRNA and are associated with
several steps of mRNA processing, sorting, and splice control.
Although most hnRNPs are nuclear resident proteins, some
can shuttle between the nucleus and the cytoplasm (16), and
they seem to execute different tasks in these compartments.
For the pyrimidine tract-binding protein (PTB/hnRNPI), which
is one of the most intensely studied proteins of the hnRNP
family, the proposed functions in the nucleus involve controlling alternative pre-mRNA splicing and thus tissue-specific
translation of a variety of different proteins (18). In addition,
the same protein seems to participate in nuclear export of
mature mRNAs, their directed transport within the cytoplasm,
and even translation and regulation of mRNA stability (11).
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FIGURE 2. During myogenesis, raver1 moves from a nuclear position to the
cell periphery. Double-fluorescence images obtained with antibodies against
raver1 and the transcription factor myocyte enhancer factor (MEF)-2 reveal
that, in undifferentiated mouse C2C12 myoblasts (MB), both proteins reside
exclusively in the nucleus (A). After induction of differentiation and myoblast
fusion, raver1 leaves this position and is also seen in the sarcoplasma,
whereas MEF-2 stays in the nuclear compartment (B). When differentiation
has proceeded to cross-striated myotubes (MT), raver1 is seen at cytoplasmic
structures that probably correspond to costameres in the fully differentiated
muscle (cf. Fig. 3), whereas the nuclei are devoid of raver1 signals (C; at right
is shown a higher magnification from the images at left). Bars = 10 m
(Reprinted with permission from Ref. 7).
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has shed new light on the function of members of this protein
family, showing that their catalog of functions can still be
larger. The sequence of the 80-kDa polypeptide of raver1 displays three NH2-terminally located RRMs with homology to
other members of the hnRNP family, in particular
PTB/hnRNPI, two bona fide nuclear location sequences (NLS),
and one REV protein-like nuclear export sequence (NES).
These motifs are indeed considered a telltale for an hnRNP
protein, and it was shown in vitro that raver1 can also form
heterocomplexes with other hnRNPs, for example with
PTB/hnRNPI (7). In many different cell types, raver1 was found
located in the nucleus, with a high local concentration in morphologically discrete nuclear particles. In heterokaryon assays
with hybrids of mouse and raver1-transfected human cells,
raver1 was seen to shuttle between the nucleus and the cytoplasm. This ability is probably based on the presence of both
active NLS and NES sequences. Shuttling of raver1 is not confined to transfected cells but also occurs in nonmanipulated
cells, where it is apparently correlated with tissue differentiation: in the nucleus of undifferentiated myoblasts, raver1 colocalizes with PTB/hnRNPI in the perinucleolar bodies (Fig. 1).
These are small, distinct structures adjacent to nucleoli that
are enriched in hnRNPs and believed to participate in the generation of mRNAs (17). It is probably in this cellular region that
raver1 interacts with PTB/hnRNPI and exerts its hnRNP-like
activity; preliminary data from an in vitro assay indicate that
raver1 contributes as a coregulator to alternative splicing of actinin pre-mRNA as induced by PTB/hnRNPI (N. Gromak, S.
Hüttelmaier, and C. Smith, personal communication). During
myogenesis, raver1 starts migration; it changes its location
from a purely nuclear position in myoblasts to the presumptive costameres of differentiating myotubes (Fig. 2). Finally, in
mature muscle, raver1 is associated with microfilament membrane attachment sites, such as costameres of skeletal muscle
(Fig. 3; Ref. 7) and the intercalated disks of cardiomyocytes (S.
Illenberger and B. M. Jockusch, unpublished observations).
How is raver1 targeted to these sites? In a yeast two-hybrid
analysis, raver1 was identified as a direct ligand for three
structural proteins of the intercalated disk: metavinculin, vinculin, and -actinin. Whereas metavinculin, a larger-splice
form of vinculin, is only expressed in smooth, skeletal, and
cardiac muscle [in the latter apparently contributing to the
force transmission in the beating heart (14)], vinculin and actinin are ubiquitous structural components of adhesive
junctions. All three proteins may thus recruit raver1 to (muscular) microfilament attachment sites. Biochemical and
immunoprecipitation studies demonstrated that raver1 binds
directly to these proteins and that ternary complexes comprising raver1, vinculin, and -actinin could be precipitated from
cell lysates (7). Comparing the binding properties of the fulllength raver1 with those of appropriate deletion fragments
revealed that the NH2-terminal half of raver1 harbors the binding sites for vinculin/metavinculin, whereas the COOH-terminal half of the molecule interacts with -actinin (Fig. 4A). Furthermore, it turned out that raver1 also colocalizes with vinculin and -actinin in microfilament-plasma membrane
attachment sites of adhesion complexes in epithelial or fibroblastic cells (7). Hence, this hnRNP protein is a ubiquitous
FIGURE 3. Raver1 colocalizes with vinculin/-actinin at microfilament
attachment sites in mouse skeletal muscle. The immunofluorescence images
show frozen sections of mouse M. soleus after double staining with antibodies against raver1 (A and D), vinculin (B), and -actinin (E). The overlays (C
and F) reveal a substantial colocalization of raver1 with both of these proteins, which are structural components of costameres, the microfilament
attachment sites connecting the periphery of Z-lines with the sarcolemma.
Bars = 10 m (Reprinted with permission from Ref. 7).
component of a wide variety of adhesive structures, which
suggests that it is an important factor in their assembly.
What is the significance of raver1 targeting to microfilament-associated adhesion complexes, especially during myogenesis? Given a possible role of raver1 in mRNA transport
and targeting, it is interesting to note that vinculin mRNA was
located at costameres in embryonic muscle (13); hence, the
differentiation-associated migration of raver1 to costameres
and intercalated disks may reflect its role in vinculin mRNA
transport, but there are as yet no data on either vinculin RNA
splicing or transport.
On the basis of these data, one might postulate multiple
roles of raver1 in assembling adhesion complexes. Nuclear
residence of raver1 would be achieved by functional NLS, and
the protein might be retained in this position by tethering to
nascent transcripts, as was suggested for PTB/hnRNPI (9) and
another shuttling hnRNP, A1 (19). Once transcription and
splicing of a particular subset of mRNAs (specific for adhesion
complexes) is finished, raver1 might use its NES to leave its
position and travel to the cytoplasm, taking the corresponding
mRNAs along. Raver1, together with the spliced mRNAs,
would then be incorporated into locasomes and transported
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FIGURE 4. The putative role of raver1 in mRNA processing and translation of cell adhesion proteins. A: deduced amino acid sequence and ligand-binding motifs
of raver1. The polypeptide sequence is shown in orange. Sequence motifs with a recognized function as deduced from databases are indicated as follows: the
two nuclear location signals are shown in black, the single nuclear export signal is shown in white, and the three RNA-recognition motifs are hatched. Binding
regions for the three protein ligands identified so far are shown by double-headed arrows underneath the sequence diagram. The numbers refer to amino acids.
B: how raver1 may act as a tour guide during the formation of adhesion complexes. In the nucleus, raver1 participates in RNA processing within spliceosomes,
probably in concert with PTB/hnRNPI. Both proteins may also be part of the export complex, transporting the mature mRNA into the cytoplasm. In specialized
locasomes that have yet to be characterized in their molecular composition, raver1 together with other cofactors is transported via cytoskeletal elements to
peripheral cell adhesion sites. By binding to -actinin and vinculin, integral proteins of attachment plaques, raver1 may help anchor the mRNA to these structures and allow site-specific translation, thus contributing to the assembly of cell adhesion complexes.
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along cytoskeletal elements. Once these particles reach their
destination at the cell periphery, translation is initiated, the
protein products are tethered to raver1 in this position, and
such complexes contribute to the assembly of adhesion structures in muscle and nonmuscle cells. The model shown in Fig.
4B emphasizes the most important steps in such a process.
5.
6.
7.
Conclusions
We apologize to those authors whose publications could not be cited due
to lack of space.
Our own work referred to in this article was supported by the Deutsche
Forschungsgemeinschaft.
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14.
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16.
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