musashi: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - musashi

Synonyms -

Cytological map position - 96E1--96E4

Function - RNA-binding-protein

Keywords - central and peripheral nervous systems

Symbol - msi

FlyBase ID:FBgn0011666

Genetic map position - 3-[87]

Classification - RNP-1 and RNP-2 motifs

Cellular location - nuclear



NCBI links: Entrez Gene

musashi orthologs: Biolitmine
Recent literature
Sharma, A., Akagi, K., Pattavina, B., Wilson, K. A., Nelson, C., Watson, M., Maksoud, E., Harata, A., Ortega, M., Brem, R. B. and Kapahi, P. (2020). Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila. Sci Rep 10(1): 19080. PubMed ID: 33154387
Summary:
Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. This study developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) was performed to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, an RNA binding protein, Musashi (msi), was identified as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, this study has established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.
Siddall, N. A., Casagranda, F., Johanson, T. M., Dominado, N., Heaney, J., Sutherland, J. M., McLaughlin, E. A. and Hime, G. R. (2022). MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function. Cell Death Discov 8(1): 455. PubMed ID: 36371343
Summary:
The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. This study has identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). FSC maintenance and escort cell function were shown to specifically require the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. It was further demonstrated that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, this study shows that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.
BIOLOGICAL OVERVIEW

Adult mechanosensory bristles consist of four cells: two cells of neural derivation (neuron and glial or thecogen cells), and two support cells ( a socket cell [tormogen] and a shaft cell [trichogen]). These four cells are derived from a single precursor called the sensory organ precursor (SOP). Mutations in musashi and numb both result in an increase in the ratio of support cells, at the expense of neural cells. Mutation of a third gene, tramtrack, results in the opposite phenotype: the transformation of socket and shaft cells to neuron and glial cells. Ectopic expression of ttk has just the opposite effect: the transformation of neuron and glial cells into socket and shaft cells. Numb protein is asymmetrically distributed to neural precursor cells in the first division of the SOP, and Numb targets tramtrack, which then acts as a repressor of support cell fate in neural and glial progeny (Rhyu, 1994 and Guo, 1995). A double-shaft phenotype is abundant in musashi mutants, giving the gene its name -- typically, samurai warriors used only one sword; however, the famed artist-samurai Musashi Miyamoto (1584-1685) originated a style of fighting using two swords simultaneously (Nakamura, 1994).

Where does Musashi fit in the pathway that genetically controls mechanosensory bristle cell fate? MSI is a putative RNA binding protein. Analysis of the mutant phenotypes suggests that MSI acts at two levels. At the first level, the progeny of the SOP are distinguished or earmarked as being either neuron/glial precursors or shaft/socket precursor cells. At a second and later stage, the progeny of the shaft/socket cells are distinguished as being either shaft or socket cell precursors. Failure of Musashi action results in an excess of shaft and socket precursors at the expense of neuron and glial cells in the first instance, and an excess of shaft cells at the expense of socket cells in the second instance (Nakamura, 1994).

Because Musashi is nuclear, it is thought that it does not function to regulation translation, stability or subcellular localization of mRNA. Instead Musashi may regulate target mRNAs at the level of mRNA processing. Many nuclear RNA binding proteins bind pre-mRNA (high molecular weight nuclear RNAs also known as hnRNAs) and either facilitate or hinder the interaction of these hnRNAs with other components that are necessary for processing hnRNAs. The function of only a few RNA binding proteins are understood in great detail The best-characterized examples are the Drosophila Sex Lethal and transformer-2 proteins. It has been proposed that MSI controls sensillum development by regulating target genes posttranscriptionally (Nakamura, 1994).

It is also possible that MSI is required for processing Numb mRNA in the SOP. Equally likely is an involvement in processing Tramtrack mRNA. There are two alternatively spliced Tramtrack transcripts differing in alternative sets of zinc fingers as well as use of different polyadenylation signals (Harrison, 1990). It is possible that Musashi regulates Tramtrack pre-mRNA splicing, controlling the ratio of alternatively spliced products (Nakamura, 1994).

Expression of murine Musashi homolog, m-Msi-1, shows a pattern complementary to that of another mammalian RNA-binding protein, Hu (a mammalian homolog of the Drosophila neuron-specific RNA binding protein ELAV), which is exclusively expressed in postmitotic neurons in the CNS. It is possible that m-Msi-1 and Hu have distinct roles in neurogenesis that are relevant to those of Drosophila MSI and ELAV, respectively (Sakakibara, 1996).

Translational repression determines a neuronal potential in Drosophila asymmetric cell division

Asymmetric cell division is a fundamental strategy for generating cellular diversity during animal development. Daughter cells manifest asymmetry in their differential gene expression. Transcriptional regulation of this process has been the focus of many studies, whereas cell-type-specific 'translational' regulation has been considered to have a more minor role. During sensory organ development in Drosophila, Notch signaling directs the asymmetry between neuronal and non-neuronal lineages, and a zinc-finger transcriptional repressor Tramtrack69 (Ttk69) acts downstream of Notch as a determinant of non-neuronal identity. Repression of Ttk69 protein expression in the neuronal lineage occurs translationally rather than transcriptionally. This translational repression is achieved by a direct interaction between cis-acting sequences in the 3' untranslated region of ttk69 messenger RNA and its trans-acting repressor, the RNA-binding protein Musashi (Msi). Although msi can act downstream of Notch, Notch signaling does not affect Msi expression. Thus, Notch signaling is likely to regulate Msi activity rather than its expression. These results define cell-type-specific translational control of ttk69 by Msi as a downstream event of Notch signaling in asymmetric cell division (Okabe, 2001).

Mechanosensory bristle development in Drosophila is an excellent model system in which to address the molecular mechanisms of asymmetric cell division. Four successive asymmetric cell divisions from a common precursor cell, called the sensory organ precursor (SOP) generate a sensory bristle comprising four different non-neuronal support cells and one neuron. The first asymmetric cell division of SOP into IIa non-neuronal and IIb neuronal precursors is regulated by Notch signaling; specific activation of Notch occurs in IIa owing to inhibition in IIb by Numb, an intracellular negative regulator of Notch. Activation of Notch signaling results in the appearance of Ttk69 protein in the IIa precursor, but not in IIb. The expression pattern of Ttk69, phenotypes of ttk69 loss-of-function mutants, and the overexpression of Ttk69 suggest that Ttk69 is necessary and sufficient to specify the IIa non-neuronal lineage; however, the mechanism through which Notch signaling regulates Ttk69 expression has remained elusive (Okabe, 2001).

Although Ttk69 protein is not detected in the IIb neuronal precursor in which Notch signaling is inactive, similar levels of ttk69 mRNA are present in the IIb precursor and its non-neuronal sibling. Using a lacZ reporter in a ttk enhancer trap line, it has been confirmed that there are indistinguishable levels of expression between IIa and IIb. Selective repression of Ttk69 protein expression in the IIb neuronal precursor thus must occur post-transcriptionally, either by post-translational degradation or translational repression (Okabe, 2001).

Ttk69 protein also regulates neural development in developing adult photoreceptor cells, where Ttk69 expression is controlled by selective degradation in neuronal cells dependent on seven in absentia (sina) and phyllopod functions. These two genes are also involved in the cell-fate decision of mechanosensory bristle lineage; loss of either gene function causes a 'double-bristle phenotype', suggesting that the IIb precursor is transformed into IIa. However, as the penetrance of the sina-null mutant phenotype (12.1% transformation of IIb precursor into IIa) is much less than that of the phenotype of overexpression of ttk69, other mechanisms must exist for post-transcriptionally regulating ttk69 (Okabe, 2001).

A good candidate for a repressor of ttk69 is the msi gene product. Loss of msi function causes a double-bristle phenotype similar to that of overexpression of Ttk69 (36.3%). Furthermore, it was found that in msi mutants Ttk69 protein is ectopically expressed in IIb precursors, and that a reduction of one copy of the ttk69 gene dominantly suppresses the msi double-bristle phenotype (14.3%). Loss of ttk transforms IIa precursor into IIb precursor even in the absence of msi, confirming that ttk69 acts downstream of msi in the first asymmetric cell division (Okabe, 2001).

These results indicate that the msi phenotype is caused by a derepression of Ttk69 protein expression in the IIb neuronal precursor. This msi-dependent pathway acts parallel to the sina-dependent pathway, because a sina;msi double-null mutation shows a much more severe phenotype than either one alone (the production of extra outer support cells in 95.3% of bristles. Since msi encodes an RNA-binding protein containing two RNA recognition motifs, Msi may act directly on cis-acting sequences of the ttk69 mRNA and regulate expression of Ttk69 protein at the translational level (Okabe, 2001).

To identify cis-element(s) crucial for the post-transcriptional regulation of ttk69 mRNA, advantage was taken of ttk1, an allele of ttk with a transposable element (P-element) insertion in the 3' untranslated region (UTR) of ttk69 mRNA. Although this insertion causes the loss of another isoform of TTK (Ttk88), it also behaves as a gain-of-function allele of ttk69; Ttk69 protein is ectopically expressed in the IIb precursor in ttk1 pupal nota. In fact, it was found that ttk1 mutants display a semi-dominant double-bristle phenotype (5.3% in heterozygous mutants; 26.2% in homozygous mutants), a phenotype identical to overexpression of either Ttk69 or Ttk88. Thus, the bristle phenotype in ttk is caused by ectopic expression of Ttk69 rather than the loss of Ttk88 (Okabe, 2001).

Analysis of the ttk69 cDNA from ttk1 reveals that its ttk69 mRNA lacks the distal 80% of the 3' UTR sequence, owing to a read-through into P-element sequences. These results indicate the importance of the 3' UTR of ttk69 mRNA in vivo for preventing Ttk69 protein expression in the IIb precursor. Thus, the absence of msi or the ttk69 mRNA 3' UTR similarly results in ectopic expression of Ttk69 protein in the IIb precursor and the double-bristle phenotype (Okabe, 2001).

To test whether Msi binds ttk69 mRNA directly, Msi target sequences in vitro were analyzed in vitro, and these sequences were sought in the ttk69 mRNA. Random 30- or 50-base-pair RNA polymers were selected by using recombinant gluthathione S-transferase (GST)-Msi fusion protein immobilized to glutathione beads, and candidate RNAs were concentrated by five cycles of polymerase chain reaction with reverse transcription (RT-PCR) and in vitro transcription. 48 independent RNA polymers were isolated and sequenced, and it was found that all contained a common motif of GU3-6(G/AG) (Okabe, 2001).

Fifteen such sequences are present in the 3' UTR of the ttk69 mRNA, and ten of these are located in the region that is absent in the ttk69 mRNA produced in the ttk1 mutant. By gel mobility shift assays, it has been shown that recombinant Msi protein binds specifically to these sequences in ttk69 mRNA. The RNA ß-region, containing three Msi target sequences (corresponding to position 3,871-3,880 of ttk69 cDNA), was bound by Msi but not by a mutant Msi protein with substitutions in the RNA recognition motifs (MsiA*B*). Binding of the RNA ß-region can be competed by a homologous sequence, but not by a region of ttk69 mRNA lacking Msi-binding motifs. These results show that Msi is a sequence-specific RNA-binding protein that binds the 3' UTR of ttk69 mRNA in vitro (Okabe, 2001).

To address the functional significance of the binding of Msi to ttk69 mRNA 3' UTR, a translational reporter assay was performed in Drosophila S2 cells. Luciferase reporter genes that contained the entire 3' UTR of ttk69 (luc-ttkUTR), the proximal 20% of the 3' UTR (luc-ttkUTRdelta) or that of alcohol dehydrogenease gene (luc-AdhUTR) were introduced in S2 cells that express Msi, and translational and transcriptional outputs were quantified by measuring the enzymatic activities and mRNA levels (Okabe, 2001).

In cells expressing Msi, these three reporter genes produce roughly the same amount of mRNA. When the translational output was measured by luciferase activity, however, the luc-ttkUTR reporter produced only 24.7% activity as compared with the Adh 3'UTR. The luc-ttkUTRdelta, which has 5 of the 15 Msi-binding sites present in the ttk69 3' UTR and mimics ttk69 mRNA produced in ttk1, exhibits only a modest decrease in the translational output (69.8% activity as compared with luc-AdhUTR). This is consistent with the sharp reduction in Msi-dependent translational repression in the ttk1 mutant, because the penetrance of ttk1 homozygous is less than that of msi null homozygous. The effect of ttk69 3' UTR is dependent on the RNA-binding activity of Msi; in cells expressing Msi lacking RNA-binding activity (MsiA*B*), translational repression of the ttk69 3' UTR is not observed. The 3' UTR of ttk69 mRNA therefore confers Msi-dependent translational repression in this assay system. Together, these results indicate that Msi inhibits translation of ttk69 mRNA in IIb precursors by binding to its 3' UTR (Okabe, 2001).

Although the translational inhibitory effect of Msi on ttk69 mRNA is specific to the IIb precursors, Msi protein is present in both IIa and IIb precursors. Thus, IIa precursors must somehow be able to escape the action of Msi as a translational repressor of Ttk69, probably through the effect of Notch signaling. Loss of Notch function in the SOP lineage causes the transformation of IIa precursor into IIb, with mutants showing a balding phenotype owing to loss of socket and shaft cells. This phenotype in IIa precursor is dependent on Msi activity; loss of both Notch and msi function results in a dense double-bristle phenotype with no neurons in the subepidermal layer, indicating that the IIb precursor took the non-neuronal fate. msi is thus epistatic to Notch during the asymmetric cell division giving rise to IIa and IIb precursors. Taken together, it is proposed that Notch inhibits Msi activity in the IIa precursor (non-neuronal cell), thus allowing translation of ttk69 mRNA, whereas in the IIb precursor (neuronal cell), where Notch is inactivated by Numb, Msi prevents ttk69 translation (Okabe, 2001).


GENE STRUCTURE

cDNA clone length - 3.7 kb

Bases in 5' UTR - 251

Exons - 3

Bases in 3' UTR - 1666


PROTEIN STRUCTURE

Amino Acids - 606

Structural Domains

MSI is homologous to RNA-binding proteins (RBPs). Many RBPs contain a conserved sequence of 80-90 amino acids, termed an RNA-binding domain (RBD) that includes two short highly conserved motifs called RNP-1 and RNP-2. The MSI protein contains two RBDs (each with two RNPs). One of the gene products most homologous to MSI is a Xenopus nervous system-specific putative RBP, NRP-1, as well as another Xenopus putative RBP, XRP-1. Each is 60% identical to MSI in the RBDs. MSI also contains amino acids in the RBDs about 50% and 42% identical (respectively) to two heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins: human hnRNP A/B protein and Drosophila HRP-40. The two MSI RBDs differ significantly in sequence. Outside of RNP-1 and RNP-2, the two RBDs are only 33% identical to one another. MSI contains two regions of 85 and 179 amino acids that are rich (about 45%) in alanine and glutamine residues. Alanine/glutamine-rich regions are also present in the Drosophila nervous system-enriched putative RBPs: ELAV and Couch potato (Nakamura, 1994).


musashi: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 12 December 2022 

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