1 Department of Physiology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan
2 Department of Developmental Genetics, National Institute of Genetics, and
Department of Genetics, Graduate University for Advanced Studies, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan
* Author for correspondence hidokano{at}sc.itc.keio.ac.jp
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Summary |
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Key words: Translational regulation, Cell fate, Musashi, RNA-binding protein, Asymmetric cell division, Neural stem cell, Notch signaling
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Introduction |
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Translational regulation of sexual identity in C. elegans |
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Translational regulation during sex-specific Drosophila development |
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Musashi and translational control of asymmetric cell division in Drosophila |
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The Drosophila external sensory organ is an excellent model system
for studies of mechanisms that regulate asymmetric cell divisions during
development (Jan and Jan,
1998). Previously, we identified a neural RNA-binding protein,
Musashi (MSI), that is required for the asymmetric cell division of the
sensory organ precursor cell (SOP) of the Drosophila adult external
sensory organ (Nakamura et al.,
1994
). In the wild-type fly, the SOP generates two second-order
precursor cells: a non-neuronal precursor cell (the IIa cell) and a neuronal
precursor cell (the IIb cell). In contrast, in the loss-of-function
msi mutants, the SOP fails to undergo asymmetric cell division and
instead gives rise to two IIa cells. Consequently, the number of socket and/or
shaft cells is increased at the expense of neurons and glia, which results in
a `double bristle'
phenotype*
(Fig. 1). Molecular analysis of
the msi locus demonstrated that it encodes an RNA-binding protein
that has two tandem RNA-recognition motifs (RRM-1 and RRM-2), each of which
includes two short, highly conserved motifs: RNP-1 (eight residues) and RNP-2
(six residues) (reviewed by Dreyfuss et
al., 1993
). This structure led us to propose that
post-transcriptional control plays a role in this asymmetric cell division.
However, the precise role of MSI in this process remained obscure, mainly
because of the difficulties of determining its in vivo target RNA.
|
Intensive biochemical and genetic studies demonstrated that the in vivo
target of MSI is the tramtrack69 (ttk69) mRNA, which encodes
the key determinant of IIa versus IIb fate
(Okabe et al., 2001). The
TTK69 protein is a zinc-finger-type transcriptional repressor, whose
expression is necessary and sufficient to specify a non-neuronal identity
(reviewed by Jan and Jan,
1998
). The regulatory mechanism that ensures that TTK69 protein is
present only in the IIa cell was unknown. Surprisingly, we found that
ttk69 mRNA is expressed in both IIa and IIb cells at apparently equal
levels, which indicated that the synthesis of the TTK69 protein is regulated
translationally rather than transcriptionally. The underlying molecular
mechanism was revealed by intensive analysis of a gain-of-function allele of
ttk (ttk1)
(Xiong and Montell, 1993
). The
ttk1 mutants possess a P-element insertion in the 3'
UTR of the ttk69 mRNA and ectopically express TTK69 protein in the
presumptive IIb cell. This suggested that the cell-type-specific translational
repression of TTK69 depends on cis-acting repressor sequences in the
ttk69 3' UTR. Subsequently, in vitro selection experiments
(Buckanovich and Darnell, 1997
)
and biochemical assays showed that MSI protein specifically binds to
cis-acting repressor RNA sequences that contain GU3-6[G/AG] repeats
in the 3' UTR of ttk69 mRNA to execute its translational
repression. Indeed, in the msi mutant, the translation of
ttk69 mRNA is de-repressed and the TTK69 protein is ectopically
produced in the presumptive IIb cell (Fig.
2) in a way similar to that seen in the gain-of-function
ttk1 mutant. Consequently, the presumptive IIb cell could
have transformed into a IIa cell, thus producing double-bristle phenotype.
Although the molecular mechanism responsible for the absence of MSI function
in the IIa precursor cells remains to be elucidated, it is possible that the
function of MSI is regulated post-translationally by Notch signalling, which
is differentially activated in IIa precursor and IIb precursor cells
(Okabe et al., 2001
).
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Roles of the Musashi family in the mammalian nervous system |
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The selective expression of Musashi1 in stem cells or immature cells of these tissues led us to speculate that it plays a role in keeping these cells in an undifferentiated state during post-transcriptional gene regulation. We sought to identify its target RNA by using a strategy similar to that used in the study of Drosophila Musashi. By in vitro selection, we determined that the consensus ligand RNA sequence for mammalian Musashi1 is G/AU2-3(AGU). We then explored candidates for the in vivo Musashi1 target gene on the basis of the results of in vitro selection experiments as well as expression patterns and functions. We speculated that mRNAs of genes regulating neural differentiation (either positively or negatively) would be downstream targets of Musashi1 since Musashi1 is preferentially expressed in undifferentiated neuronal progenitor cells.
One of the in vivo targets of Musashi1 is m-Numb mRNA, the
3' UTR of which has a Musashi1-binding site
(Imai et al., 2001). The
m-Numb and Musashi1 expression patterns overlap in neuroepithelial
cells in the ventricular zone of the neural tube
(Sakakibara et al., 1996
;
Zhong et al., 1996
;
Zhong et al., 1997
).
Furthermore, m-Numb is involved in the regulation of neuronal
differentiation (Wakamatsu et al.,
1999
). Studies using both gain-of-function and loss-of-function
mutations have demonstrated that Musashi1 translationally represses synthesis
of m-Numb. Because Numb is an evolutionarily conserved intracellular Notch
antagonist (Uemura et al.,
1989
; Guo et al.,
1996
; Zhong et al.,
1996
; Zhong et al.,
1997
), we expected Musashi1 to be a positive regulator of Notch1
signaling (Fig. 3). Indeed,
overexpression of Musashi1 activates Notch1 signaling through a pathway
dependent on the action of RBP-J
(Imai et al., 2001
), a dormant
transcription factor of the CSL family that forms a functional complex with an
intracellular domain of Notch1 protein within the nucleus
(Schroeder et al., 1998
).
Notch signaling is known to induce the self-renewal of mammalian neural stem
cells (Nakamura et al., 2000
;
Gaiano and Fishell, 2000
). By
reducing the activity of mammalian musashi genes [including those
encoding Musashi1 and Musashi2 (Sakakibara
et al., 2001
)] through the antisense ablation of Musashi2 protein
production in cultured brain cells derived from
musashi1-/- mice, we found that these genes play essential
roles in maintaining the undifferentiated state (or self-renewal) of neural
stem cells (S. Sakakibara and H. Okano, unpublished).
|
Musashi1 is expressed in particular types of brain tumor that are likely to
have originated from immature brain cells
(Toda et al., 2001;
Kanemura et al., 2001
).
Interestingly, the Musashi1 expression level correlates with the malignancy
and proliferative activity of the tumor. Furthermore, glioblastoma cells that
express high levels of Musashi1 show a significantly higher rate of nuclear
localization (and hence activation) of Notch1 than do cells expressing lower
levels of Musashi1 (Kanemura et al.,
2001
). Cells that stained strongly for Musashi1 showed almost no
staining for m-Numb. Thus, a high level of Musashi1 expression could have led
to the clonal expansion of the above-mentioned tumor cells by activating Notch
signaling, presumably through the translational inhibition of m-Numb
(Kanemura et al., 2001
).
Hyperactivation of Notch signaling could result in tumorigenesis, possibly
owing to the inhibition of apoptosis and maintenance of a sustained immature
state (Artavanis-Tsakonas et al.,
1999
). The trigger causing Musashi1 overexpression, which could be
the initial step in tumorigenesis, remains to be elucidated. Nevertheless, it
is clear that members of the Musashi family are involved in determining cell
fate not only during normal neural development but also in a particular
pathogenic state.
The molecular mechanism underpinning repression of the translation of m-Numb mRNA by Musashi1 remains to be elucidated. However, in the case of TRA, a single RNA-binding protein could act as a multifunctional regulator that controls its target genes at several different steps of post-transcriptional regulation, including splicing, translation, stability control and localization of RNAs. However, why a single RNA-binding protein might have a multifunctional role in post-transcriptional gene regulation remains an open question. Interestingly, intracellular localization of Musashi1 protein is variable (cytoplasmic and/or nuclear), depending on the cell-type and/or developmental stage. Thus, Musashi1 could be involved at a step other than translational control something we and others are currently investigating.
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Conclusion and Perspectives |
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Acknowledgments |
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Footnotes |
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References |
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