INTRODUCTION

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Posttranscriptional regulation plays essential roles in the wide variety of events that occur during animal development, including the localization of maternal-effect gene products within oocytes, cell fate determination, and cell polarity formation by controlling alternative splicing, mRNA stability, RNA transport, and/or the translation of existing mRNAs (11, 57, 66). Among developing tissues, the nervous system in particular uses a variety of posttranscriptional means to regulate the vast cellular diversity and synaptic plasticity that is generated. Neural RNA-binding proteins are likely to play essential roles in mediating these processes (44).

Two classes of neural RNA-binding proteins with ribonucleoprotein-type RNA recognition motifs (RRMs), the Elav and Musashi subfamilies, have been proposed (44). The Elav subfamily includes the mammalian Elav homologue, Hu proteins, whose members are expressed in postmitotic neurons and have been proposed to be required for the survival or terminal differentiation of these cells (1, 2, 34, 45, 64, 69). Intensive study in this area revealed that Hu proteins regulate the gene expression involved in neuronal differentiation by controlling RNA stabilization or translation (2, 13, 35, 48, 64). On the other hand, the Musashi subfamily proteins are expressed in neural precursor cells rather than postmitotic neurons (15, 28, 44, 49, 52).

Musashi1 (Msi1) was isolated as a mammalian homologue of Drosophila Musashi (d-Msi), which is required for the asymmetric cell division of sensory neural precursor cells (38). In Drosophila, genes that are responsible for the proper asymmetric cell division of sensory organ precursor cells or central nervous system neuroblasts have been identified and extensively investigated. Interestingly, many of these genes have been shown to be involved in the regulation of Notch signaling, including numb, tramtrack, Notch, and Delta (reviewed by Jan and Jan [23]). Recently, our laboratory showed that d-Msi represses the expression of tramtrack, which encodes a transcriptional repressor, at the level of translation (21, 43). We also identified the mammalian homologue of Drosophila Musashi, Musashi1, which is highly enriched in neural progenitor cells in the developing mouse embryonic central nervous system (28, 52, 53). Msi1 expression is gradually down-regulated during the course of neural differentiation. The Msi1 protein consists of 362 amino acid (aa) residues, and it contains two conserved RRMs in its N-terminal half and a putative nuclear export signal in its C-terminal half, which is consistent with its observed localization of the cytoplasm in embryonic neural progenitor cells (28, 52).

To help elucidate the role of Msi1 protein in neural progenitor cells, we sought to determine (i) the RNA sequences that bind to Msi1, (ii) an in vivo target RNA of Msi1, and (iii) the mechanism of action of Msi1 on the expression of its downstream target genes. To this end, in the present study we identified the RNA sequence for Msi1 and demonstrated putative translational repression of a likely in vivo target gene mammalian numb (m-numb).

	MATERIALS AND METHODS

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Preparation of Musashi1 fusion protein. To prepare mouse Musashi1 fusion protein (Msi1-2TR), a plasmid vector, pET21a-msi12TR, was constructed by inserting a part of the coding region (corresponding to aa 7 to 192) of the musashi-1 cDNA into the pET21a expression vector (Novagene). The plasmid was introduced into Escherichia coli strain BL21 (DE3) pLysS. The expression and affinity purification of the fusion protein were performed as described previously (28).

Selection of Musashi-1 RNA ligands. RNA selection was basically performed according to previously described methods (6, 61). Oligonucleotides harboring a 50-bp random sequence surrounded by primer binding sites (5'-GGGAAGATCTCGACCAGAAG-N₅₀-TATGTGCGTCTACATGGATCCTCA-3') were synthesized using a DNA synthesizer (Nissinbo). The oligonucleotides were amplified by PCR using a forward primer containing the T7 promoter sequence and a reverse primer (forward primer: 5'-CGGAATTCTAATACGACTCACTATAGGGAAGATCTCGACCAGAAG-3'; reverse primer: 5'-TGAGGATCCATGTAGACGCACATA-3'). The library DNAs were transcribed in vitro with T7 RNA polymerase and [-³²P]UTP (Amersham Pharmacia Biotech). The RNA was applied to a column which was filled with nickel affinity resin preadsorbed with 100 µg of purified histidine-tagged Msi1 fusion protein, in binding buffer (0.5 M LiCl, 20 mM Tris-HCl [pH 7.5], 1 mM MgCl₂). The beads were then washed with 10 ml of binding buffer. Bound RNA was eluted from the column in elution buffer (20 mM Tris-HCl [pH 7.5], 1 M imidazol), phenol extracted, and ethanol precipitated. The RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Gibco BRL), cDNA was used for PCR with the forward and reverse primers given above under the following conditions: 15 cycles of 1 min at 94°C, 1 min at 59°C, and 1 min at 72°C. The PCR product was used for the next round in the selection procedure. This process was repeated an additional seven times before the products were subcloned into the pUC119 vector (Clontech). We predicted RNA secondary structure using the commercial sequence analysis software DNASIS (Hitachi Software Engineering Inc.) program based on the Zuker-Stiegler method.

Gel shift assays. Gel shift assays were performed with various amounts of Msi1 fusion protein in 16 µl of KNET buffer (32). Ten thousand counts per minute (approximately 4 fmol) of selected ³²P-labeled RNA ligand (S8-13 and S8-19) was added to the solutions containing Msi1 fusion protein. For the competition experiment, unlabeled RNA was added before the ³²P-labeled RNA. Protein and RNA samples were allowed to equilibrate for 30 min at room temperature. After incubation, the mixtures were immediately loaded onto 8 or 15% polyacrylamide gels (0.5× Tris-borate-EDTA buffer, 5% glycerol) and fractionated by electrophoresis. The gels were then dried and exposed to XAR autoradiography film (Kodak).

In vitro binding assay using m-numb 3' UTR. [³⁵S]methionine-labeled full-length Msi1 protein was prepared by an in vitro transcription translation system using the pRSETb-msi1 plasmid vector (52), pET21a-msi12TR, pRSETb-C17(C-terminal half), and reticulocyte lysate containing T7 RNA polymerase (Promega). The Msi1 protein was incubated with m-numb RNAs labeled with biotin-14-CTP in binding buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.05% NP-40, 0.1% sodium azide) for 30 min. The mixture of Msi1 and m-numb RNA was then added to streptavidin-agarose beads previously resuspended in binding buffer. The beads were then washed with 1 ml of binding buffer five times. The bead pellet was resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, boiled for 5 min, and spun. The supernatant was loaded onto a 15% SDS-polyacrylamide gel and fractionated by electrophoresis. After electrophoresis, the gel was dried and exposed to Fuji RX-U film at 80°C for 1.5 to 8 h.

Cell culture and in vivo binding assay. NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (Nissui) supplemented with 10% calf serum. NIH 3T3 cells were plated onto 60-mm dishes (Falcon) (10⁶ cells/dish). On the following day, cells were transfected with 1 µg of the Msi1 expression constructs (pcDNA3-FLAGMsi1HAT, pcDNA3-FLAGMsi1mutR1HAT, and pcDNA3-FLAGMsi1) shown in Fig. 4A, using the Effective transfection reagent (Qiagen). Two days later, the transfected cells were suspended in 1 ml of NET-Triton buffer (6), homogenized, and spun in a microcentrifuge. In the presence of RNase inhibitor (0.5 U/µl) (Promega), histone affinity tag (HAT)-tagged Msi1-RNA complexes in the supernatants were pulled down by Talon resin (Clontech). Extraction of precipitated RNA, DNase I treatment, and reverse transcription were performed as described previously (6). Subsequently, PCR was performed with m-numb gene-specific primers using 32 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C or with -actin-specific primers using 25 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. Primers used in the PCR were as follows: the m-numb primer set, 5'-ATGAGCAAGCAGTGTTGTCCTGG-3' and 5'-CAAGTAGCTGCAACTGGCTGG-3'; and -actin, 5'-CTTCCTCCCTGGAGAAGAGCTATGAGC-3' and 5'-GCCTAGAAGCACTTGCGGTGCACG-3'.

Reporter assay using luciferase and quantification of reporter mRNA by Northern ELISA system. NIH 3T3 cells (3 × 10⁵ cells/ml per assay) were transfected with 0.2 µg of firefly luciferase reporter vector, 20 ng of Renilla luciferase control vector pRL-TK (Toyo Ink), and 0.3 µg of pEGFP-N3 vector (Clontech) and with a combination of pcDNA3 vector (Invitrogen) and pCDNA3-T7Msi1 or pCDNA3-T7Msi1mutR1 expression vector (totaling 1.5 µg), using Fugene 6 transfection reagent (Roche). After 2 days of incubation, the cells were lysed with luciferase assay lysis buffer (Toyo Ink). The firefly luciferase (reporter) activities and Renilla luciferase activities (control) were measured with individual reaction substrate mixtures supplied by the manufacturer using a Berthold Lumat LB9507 luminometer. The ratio of reporter luciferase activity in relative light units was divided by the control Renilla luciferase activity to give a normalized reporter luciferase value.

Preparation of recombinant adenovirus and infection experiment. We generated the recombinant adenovirus Adex-FLAGMsi1 using the pAdex1pCAw vector, essentially according to previously described methods (19). The high-titer recombinant adenovirus stock (Adex-FLAGMsi1, 3 × 10¹⁰ PFU/ml; Adex- NLLacZ, 3 × 10¹⁰ PFU/ml) was obtained and stored at 80°C.

RNA quantification by Northern blot analysis. Total RNAs were extracted with Trizol reagent (Gibco BRL) from the NIH 3T3 cells infected with Adex-FLAGMsi1 described above and precipitated with ethanol. The RNAs were loaded onto morpholinepropanesulfonic acid-formaldehyde-agarose gels and then transferred to a Hybond N+ nylon membrane (Amersham Pharmacia Biotech) and probed with ³²P-labeled m-numb cDNA and -actin cDNA. Hybridization signals were detected with XAR autoradiography film (Kodak) and quantified using BAS5000 (Fuji). The ratio of hybridization signals for m-numb mRNA over those for -actin mRNA yielded normalized quantities of m-numb mRNA level. We performed two independent experiments and calculated the average value.

Sucrose gradient centrifugation. We performed sucrose gradient centrifugation as described previously (58). NIH 3T3 cells infected with Adex-FLAGMsi1 as described above were harvested by centrifugation, washed with cold phosphate-buffered saline, resuspended in buffer A (10 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol, 5 mM HEPES [pH 7.3], 2 µg of leipeptin per ml, 2 µg of pepstatin per ml, and 0.5% aprotinin), incubated on ice for 10 min, and disrupted by passage through needles. Centrifugation at 2,500 × g for 10 min yielded a pellet and a supernatant fraction designated cytoplasmic lysate. The KCl concentration was adjusted to 100 mM at this point. Cytoplasmic lysate was resolved on a linear sucrose gradient (5 to 30%) containing 100 mM KCl, 10 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol, 5 mM HEPES [pH 7.3], 2 µg of leipeptin per ml, 2 µg of pepstatin per ml, and 0.5% aprotinin. The gradients were centrifugated at 4°C in a Hitachi P40St1286 rotor at 40,000 rpm for 150 min. Following centrifugation, fractions were collected from the top of gradients (300 µl per fraction) using a Piston gradient fractionator (Biocomp, Inc.). Thirty microliters of each fraction was used for Western blotting. RNA was extracted from the fractions with phenol and precipitated with ethanol, and A₂₅₄ was measured.

HES1-promoter transactivation assay. To measure HES-1 promoter activity, NIH 3T3 cells were transfected with 0.2 µg of pHES-1p-luciferase (24) alone, together with 0.025 µg of pEF-BOS-FCDN1 (an expression plasmid for the Notch1 intracellular domain [FCDN1, aa 1747 to 2531]) (41), in combination with various amounts of pcDNA3-T7Msi1 or pEF-BOSneo-R218H (29), or in combination with 1 µg of pCDNA3-HAmNumb; 100 ng of SV40-LacZ construct or 20 ng of the Renilla luciferase control vector pRL-TK (Toyo Ink) was included in each transfection as an internal control. Three independent experiments were carried out. Bars in figures indicate the standard deviations. Luciferase activity was measured 48 h after transfection in a luminometer Lumat LB9507 (Berthold) and normalized according to -galactosidase activity or Renilla luciferase activity.

	RESULTS

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In vitro selection of high-affinity RNA ligands for Msi1. To identify the target RNA sequence of Msi1, we performed an affinity elution-based RNA selection method, SELEX (6, 32, 61). A ³²P-labeled RNA pool was synthesized in vitro using a PCR-amplified oligonucleotide library of 50-nucleotide semicomplete random sequences as templates. The synthesized RNA pool was then applied to a nickel affinity column to which Msi1 fusion protein Msi1-2TR had been absorbed previously. Msi1-2TR contained the two tandem RRM-type (8) RNA-binding domains (RBDs) (aa 17 to 192) as well as the histidine tag at its C terminus and a T7 tag at its N terminus (Fig. 1A). After being washed to remove RNAs that did not interact with the Msi1-2TR fusion protein, the Msi1-2TR fusion protein-RNA complexes were eluted in buffer containing 1 M imidazole. The elution profile of the first round of selection is shown in Fig. 1B. We monitored the elution of RNA and protein by counting the radioactivity and by performing SDS-PAGE, respectively (Fig. 1B). After each cycle, the bound RNA was extracted and reverse transcribed to the first strand of cDNA using the SELEX reverse primer (see Materials and Methods for primer sequences), and the cDNAs encoding the selected RNA sequences were amplified by PCR and used again as templates to synthesize RNAs for another cycle of binding and amplification. By repeating this affinity RNA-ligand selection, we found that the fraction of RNA binding to Msi1 increased from 0.2% in the initial RNA pool to 60% after eight selection cycles (Fig. 1C). In this way, we obtained an RNA pool that was enriched in RNA sequences that preferentially bind to Msi1.

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Selection of RNA ligands of Msi1 from a random RNA library. (A) Schematic representation of the domain structure of the authentic full-length Msi1 protein and the bacterially expressed fusion Msi1 protein, Msi1-2TR, which was used for the in vitro selection. The Msi1-2TR protein contains the two tandem RBDs of authentic Msi1 protein plus a C-terminal polyhistidine tag for affinity elution and an N-terminal T7 tag. (B) Msi1-2TR protein and bound RNAs were coeluted in the first selection column by specifically cluting the histidine-tagged Msi1-2TR fusion protein in 1.0 M imidazole. Eluted protein and RNA were detected by Coomassie brilliant blue staining and radioactivity counting, respectively. (C) Percentage of total radioactivity in loaded RNA represented by bound RNA in each selection is shown. The proportion of bound radioactivity increased to 60% after eight selection cycles. (D) Sequences of Msi1-selected RNAs. Twenty representative PCR clones encoding RNA ligands obtained after eight rounds of RNA selection are shown. The (G/A)UnAGU (n = 1 to 3) consensus sequence motif was often observed to be repeated in each selected clone. (E) Representative secondary structures of the RNA sequences selected by Msi1. Shading indicates the selected sequences. The predictions of RNA secondary structure were based on the Zuker-Stiegler method.

RNA-protein binding experiments. To further investigate whether the repeated (G/A)U_nAGU motif is an essential sequence element for the Msi1-RNA interaction, we performed binding assays using the Msi1-2TR fusion protein and RNA sequences derived from the most frequently selected clones, S8-13 and S8-19, which contain, respectively, two and three copies of sequences that match the selected consensus motif (Fig. 2A). Gel shift analysis was performed by incubating 4 fmol of labeled RNAs with various amounts of Msi1-2TR protein. Interestingly, the number of retarded bands in each experiment corresponded to the number of the sequences that matched the consensus sequence motif, (G/A)U_nAGU, within a selected clone. S8-13 RNA contains two consensus motifs, and S8-19 RNA has three motifs. Msi1 protein did not recognize the RNA-designated NC-4, which does not contain the selected consensus sequence (Fig. 2A). To examine whether the Msi1 protein bound specifically to the selected RNA, we performed competitive binding assays with unlabeled RNA containing the Msi1 selected-consensus sequence or a nonspecific competitor that did not contain the full consensus sequence (Fig. 2B). Four femtomoles of ³²P-labeled RNA (S8-13 or S8-19) was incubated with 100 fmol of Msi1 protein and a 10-, 100-, or 1,000-fold excess of unlabeled cold RNA, followed by gel shift analysis (Fig. 2B, lanes 13 to 15, lanes 18 to 20, lanes 23 to 25, and lanes 28 to 30, respectively). The intensities of the retarded bands representing the protein-RNA complex were decreased by the addition of excess unlabeled RNA containing the Msi1 recognition sequence (i.e., the same sequence as the labeled RNA) as a specific competitor. However, the intensities were not decreased by the addition of RNA that did not contain the Msi1 recognition sequence (NC-4). These observations indicated that the Msi1 protein specifically recognized the RNAs containing the sequence that matched the consensus sequences selected in vitro. The binding affinities of the selected RNA sequences to Msi1 were determined based on the intensity of the retarded band representing the RNA-Msi1 complex in the gel retardation assays. The dissociation constant K_d is equal to the protein concentration at which 50% of RNA is bound. In Fig. 2A, lane 4 and lane 9, 50% of RNA was bound to protein as determined by densitometry evaluation. K_d was calculated to be ~4 nM for S8-13 and S8-19. Thus, Msi1 was shown to bind to the RNA containing the sequences that match the consensus sequence motifs with high affinity.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   The sequence motif indicated by the in vitro selection is essential for the specific RNA binding of Msi1. (A) Gel shift assays of Msi1 protein. Msi1-2TR fusion protein binds to selected RNA (S8-13, S8-19; see Fig 1D). Four femtomoles of 32P-labeled RNA was incubated with various amounts of Msi1-2TR protein: 0 fmol (lanes 1, 6, and 11), 1 fmol (lanes 2, 7, and 12), 10 fmol (lanes 3, 8, and 13), 100 fmol (lanes 4, 9, and 14), and 1,000 fmol (lanes 5, 10, and 15), and the complexes were run on nondenaturing polyacrylamide gels. The band representing free RNA is shown with an arrow, and the retarded band is indicated with an arrowhead (also in panel B). Notably, no interaction was detected between the Msi1 fusion protein and NC4 RNA, which lacks the selected consensus sequence motif. (B) Competitive RNA-binding experiments using unlabeled RNAs. Four femtomoles of labeled RNA was incubated without (lanes 16, 21, 26, and 31) or with 10 fmol of Msi1-2TR protein and the following amounts of unlabeled RNA: 0 fmol (lanes 16, 21, 26, and 31), 0 fmol (lanes 17, 22, 27, and 32), 40 fmol (lanes 18, 23, 28, and 33), 400 fmol (lanes 19, 24, 29, and 34), and 4,000 fmol (lanes 20, 25, 30, and 35).

Msi1 binds to m-numb mRNA both in vitro and in vivo. Candidates for downstream target genes of the Msi1 protein were explored based on the results of the in vitro selection experiments. Since Msi1 is preferentially expressed in undifferentiated neuronal progenitor cells, mRNAs of genes regulating neural differentiation (either positively or negatively) may be possible downstream targets of Msi1. Then, m-numb, which encodes a Notch antagonist (14, 18, 65), is a likely candidate for an Msi1 target gene, based on the following facts. First, the 3'-untranslated region (UTR) of m-numb mRNA contains the consensus sequence motif for Msi1 binding. Second, the region of m-numb gene expression overlaps that of msi1 expression in neuroepithelial cells in the ventricular zone of the neural tube (52, 65, 70, 71). Third, m-numb is involved in the regulation of neuronal differentiation (12, 65, 72; Tokunaga et al., unpublished results).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Msi1 protein binds to the 3'-UTR of numb mRNA in vitro. (A) Structure of the numb gene according to a previous report (70). Arrows (N1, N2, and N3) indicate the regions that were individually transcribed in vitro. The vertical arrowhead indicates the region containing a putative Msi1-binding sequence (UAGGUAGUAGUUUUA). (B) Binding assays of the Msi1 protein to various transcripts from the 3'-UTR of m-numb mRNA. 35S-labeled full-length Msi1 protein (F) was pulled down with biotin-labeled N2 m-numb RNA-conjugated streptavidin-agarose beads and then the protein-RNA interaction was visualized by autoradiography. The other portions of m-numb RNA, N1 and N3, were not significantly bound by Msi1 protein. In lane , the absence of biotin-labeled RNA never caused coprecipitation of Msi1. The rectangle on the right shows the amount of total protein included per assay. (C) Schematic representation of Msi1 fusion proteins. F, full-length mouse Msi1; R, truncated protein containing two tandem RBDs (Msi1-2TR); C, truncated protein containing C-terminal potion of mouse Msi1. (D) Both full-length Msi1 protein (F) and truncated Msi1-2TR protein (R, used for SELEX) bind N2 RNA which contains the selected Msi1-binding sequence. The truncated protein containing two RNA-binding domains (R) conserves RNA-recognition specificity similar to that of the full-length Msi1 protein. The C-terminal half of Msi1 protein (C) did not bind N2 RNA. (E) The putative Msi1 binding site in 3'-UTR of m-numb mRNA is located in the loop portion of a predicted stem-loop structure. The predictions of RNA secondary structure were based on the Zuker-Stiegler method.

-actin

-actin

View larger version (37K): [in this window] [in a new window]   FIG. 4.   In vivo binding of Msi1 to m-numb RNA. (A) Schematic diagrams of Msi1 proteins FLAG-Msi1-HAT (H), FLAG-Msi1mutR1-HAT (A), and FLAG-Msi1 (F). The HAT tag at the C-terminal end is an affinity tag for Talon resin (Clontech). FLAG-Msi1mutR1-HAT is a non-RNA-binding form of Msi1 with amino acid replacements in the N-terminal RNA-binding domain. (B) Expression of Msi1 proteins H, A, and F in NIH 3T3 cells and affinity precipitation using HAT-tag were analyzed by immunoblotting with anti-FLAG monoclonal antibody. (C) In vivo RNA-binding assay combining affinity precipitation with RT-PCR. The expression of exogenous Msi1 proteins in NIH 3T3 cells was induced by transient transfection. RNA present in the precipitates with Talon resin was detected by RT-PCR amplification with the indicated primer sets. The RT () lanes showing that no amplification was seen without reverse transcriptase activity were controls to ensure that the RT-PCR was RNA dependent. The right panels indicate the amplification control experiment to ensure primer fidelity using the RT product from the initial extract before incubation with affinity resin. Lanes H, FLAG-Msi1-HAT; F, FLAG-Msi1; A, FLAG-Msi1mutR1-HAT.

Down-regulation of m-numb expression by Msi1: endogenous m-Numb expression and reporter assay. To examine the effect of Msi1 protein on the expression of endogenous m-Numb protein, we misexpressed Msi1 in NIH 3T3 cells using a recombinant adenovirus vector (Fig. 5A and B). NIH 3T3 cells were infected with Adex-FLAGMsi1 or Adex-NLlacZ adenovirus (19) under conditions that are not toxic to the cells (19). Infection with the Adex-FLAGMsi1 vector resulted in the expression of large amounts of FLAG-tagged Msi1 protein under the regulation of the CAG promoter (40), which is a modified chicken -actin promoter with the cytomegalovirus (CMV) 1E enhancer. Since Msi1 expression did not alter the expression level of tubulin, we used tubulin as an internal control to assess the effect of Msi1 on the expression level of m-Numb protein. Msi1 overexpression decreased the level of the endogenous m-Numb protein to 32% of the level in control cells which expressed LacZ by infection with Adex-NLlacZ (19) (Fig. 5A and B). However, the endogenous m-numb mRNA level remained unchanged in spite of the misexpression of Msi1 and LacZ (Fig. 5A and B). Based on these results, Msi1 protein is likely to be involved in the translational repression of m-Numb protein expression.

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Posttranscriptional down-regulation of m-numb gene expression by Msi1. (A) Recombinant adenovirus-mediated Msi1-misexpression, immunoblot analyses of m-Numb protein, and Northern blotting analysis. The infection with FLAG-Msi1-expressing adenovirus (Adex-FLAGMsi1 lane) resulted in a decrease of the endogenous expression level of m-Numb protein in NIH 3T3 cells, but the expression of LacZ (Adex NLLacZ lane) (19) and no treatment (control lane) did not. Immunoblot analyses using antitubulin monoclonal antibody and anti-FLAG monoclonal antibody were performed as control experiments. In addition, m-numb and -actin mRNAs were detected by Northern blotting analysis. (B) Relative amounts of m-Numb protein (blank bar) and m-numb mRNA (filled bar). Msi1 overexpression in NIH 3T3 cells by adenovirus vectors caused a decrease in endogenous m-Numb protein to 32% of the level in control cells expressing LacZ or 28% of the level in untreated cells (control[]). However, Msi1 overexpression did not affect the m-numb mRNA level in Adex-FLAGMsi1-infected cells compared with that in Adex-NLLacZ-infected cells or untreated cells (control[]). (C) Schematic representation of Msi1 effector and reporter constructs containing the 3'-UTR of m-numb. , pcDNA3-T7msi1; , pcDNA3-T7msi1mutR1 ( and  were under the control of the CMV promoter); a, pGVP2-numb3'-UTR; b, pGV-P2; c, pGVP2-reversednumb3'-UTR (a, b, and c were under the control of the simian virus 40 promoter). (D) Luciferase reporter assay. NIH 3T3 cells were transiently transfected in the indicated combinations with luciferase reporter constructs and increasing concentrations of pcDNA3-T7msi1 or pcDNA3-T7msi1mutR1 effector constructs. The graph depicts the dose-response relationship between exogenously expressed T7Msi1 and the luciferase activity obtained. The results are presented as ratios of firefly luciferase reporter activity over sea pansy (Renilla) luciferase activity, the latter being used as a control (means and standard errors of the means of values from three or four independent experiments). (E) Relative levels of reporter mRNAs quantified by Northern ELISA. The mRNA level of the transcript of EGFP, which does not contain the Msi1-binding site on its mRNA, was used as an internal control. The results were given as the ratios (percentages of control values) of luciferase-numb 3'-UTR chimeric mRNA content to EGFP mRNA content (means and standard errors of the means of results from three independent experiments) and relative doses of T7Msi1 are shown below the panel. The relative amount of luciferase reporter RNA containing the 3'-UTR of m-numb was not affected by increasing doses of exogenous T7Msi1. (F) Sucrose gradient profile of Msi1 protein containing particles in the cytoplasmic fraction of NIH 3T3 cells. Cytoplasmic extracts of NIH 3T3 cells infected with Adex-FLAGMsi1 with Mg2+ were subjected to zone centrifugation through 5 to 30% linear sucrose gradients in a Hitachi P40ST1286 rotor at 40,000 rpm for 2.5 h and fractionated. The curve shows the A254 of each fraction, and the positions of 40S, 60S, and 80S ribosomal particles and polysomes are indicated. The lower panel shows immunodetection analysis of FLAG-Msi1 protein using anti-FLAG monoclonal antibody.

Msi1 potentiates Notch signaling activity. To examine the biological significance of the translational repression of m-numb by the Msi1 protein, we performed a luciferase reporter assay using the HES1 promoter. The minimal HES1 promoter sequence, which has two RBP-J binding sites, is transactivated when Notch signaling is induced (5, 24, 54). Transfection of Msi1 resulted in a slight increase of HES1 promoter activity (5.1-fold activation from the basal level) (Fig. 6A). This slight up-regulation by Msi1 is likely to be attributed to the activation of endogenous Notch. We examined how the transactivating activity of the Notch1 intracellular domain (FCDN1), which is a dominant active form of Notch1 (41), was modified by transfecting exogenous Msi1 into NIH 3T3 cells. Expression of the Notch1 active form alone resulted in a 24.5-fold activation of the HES1 promoter from the basal level (Fig. 6 A). This activation is inhibited by the expression of RBP-J dominant negative form (R218H, designated DN-RBP-J in Fig. 6), which has no binding site to the target DNA and blocks the activation of Notch signal (10, 29). Furthermore, when exogenous Msi1 expression was induced together with the Notch1 dominant active form, the HES1 promoter activity was up-regulated another 2.7-fold over the activation caused by the dominant active Notch1 alone (66-fold activation from the basal level) (Fig. 6A). We found that the expression of Msi1 potentiated the HES1 promoter activity synergistically with that of dominant active Notch1 (Fig. 6A). The potentiation of HES1 promoter luciferase reporter activity by Msi1 is also suppressed by the expression of DN-RBP-J (Fig. 6A). Consequently, the induction of the HES1 promoter by Msi1 is likely to be due to the activation of Notch signaling through the RBP-J-dependent pathway. On the other hand, we found that transactivation of the HES1 promoter by Notch1 is inhibited by misexpression of m-Numb protein (Fig. 6B). Taken together, we demonstrated that misexpression of Msi1 in NIH 3T3 cells decreased the endogenous m-Numb protein level without affecting the mRNA level (Fig. 5A and B) and that m-Numb acts as an antagonist of Notch signaling in NIH 3T3 cells (Fig. 6B). Thus, Msi1 is likely to be involved in activation of Notch-signaling through the RBP-J-dependent pathway by the translational repression of m-Numb.

View larger version (58K): [in this window] [in a new window]   FIG. 6.   Msi1 expression potentiates Notch1-mediated activation of the HES1 promoter. (A) Transactivation of pHES1-Luc in NIH 3T3 cells. NIH 3T3 cells were transfected with HES1 luciferase reporter plasmids as indicated. The firefly luciferase activities, which represent the transactivation of the HES1 promoter, were determined and normalized against Renilla luciferase activity measurements (means and standard errors of the means of results of three independent experiments). (B) Expression of m-Numb protein inhibits the transcriptional activity of the HES1 promoter which is potentiated by the constitutive active form of Notch1. When the Notch1 active form is expressed in NIH 3T3 cells, the HES1 promoter is activated 24.6-fold from basal level. Cotransfection of HA-tagged m-Numb with the active form of Notch1 caused a decrease in HES1 promoter activity to 37% of the level in cells expressing the active form of Notch1 alone. Therefore, m-Numb antagonistically acts against Notch1 in NIH 3T3 cells. The firefly luciferase activities, which represent the transactivation of the HES1 promoter, were determined and normalized against Renilla luciferase activity measurements (means and standard errors of the means of results of three independent experiments). (C) Model of Msi1 function in the regulation of the Notch signal. Msi1 translationally regulates the m-numb gene expression. Since m-Numb blocks the Notch signal activation (cleavage and/or nuclear translocation with RBP-J, etc.) induced by Notch ligands (Delta, Jagged) which are expressed in neighboring cells, translational repression of m-Numb by Msi1 stimulates Notch signaling through the Notch1, RBP-J, and HES1 pathways. This potentiation of the Notch signal by Msi1 should maintain the immature proliferating status of cells expressing Msi1. The vertical arrowhead shows the Notch1 intracellular cleavage site.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Msi1 is a sequence-specific RNA-binding protein. To investigate the functions of RNA-binding proteins, it is important to identify the target sequences (or target genes). In this study, we used an in vitro selection method (SELEX) to identify high-affinity binding sequences for Msi1 from a random RNA pool. We successfully identified sequence-specific uridine-rich RNAs that bind Msi1. All the selected RNAs contained the (G/A)U_nAGU sequence motif (n = 1 to 3), and these motifs frequently appeared as two or three tandem repeats. The RNA-binding specificities of other RNA-binding proteins, including those of the RRM type, have been determined using SELEX or other in vitro selection approaches. hnRNP A1 specifically binds to RNA containing the sequence UAGGGA/U (7). hnRNP D binds to RNA containing UUAG (27). The recognition sequence for HuB (Hel-N1) contains short uridine-rich stretches, like AUUUA, GUUUA, and CUUUA (32). The KH-type RNA-binding protein, Nova-1, interacts with RNA containing three repeats of the sequence UCAU(N) (6). The selected consensus sequence motif for Msi1 is similar to those of hnRNP A1 and HuB in its inclusion of UAG and a short uridylate stretch, respectively. Interestingly, the uridine-rich Msi1-binding site is likely to be recognized by Hu proteins also, including HuB. The selected consensus sequences for Msi1, however, are distinguishable from any that have been previously reported for other RNA-binding proteins.

Putative target gene of the Msi1 protein. We proposed previously that Msi1 is involved in maintaining the undifferentiated state of neural stem cells through the posttranscriptional control of downstream genes (43, 52). To identify possible target genes of the Msi1 protein, we explored a number of genes responsible for the generation of cell diversity, neuronal cell differentiation, and other neural stem cell characteristics. One of the most likely candidate genes appeared to be m-numb (70). Its Drosophila homologue, numb (62), is required for the asymmetric cell division of the neural precursor cells of the external sensory organ and acts through the inhibition of Notch signaling (18). Recently, we and others have performed functional analysis of the vertebrate Numb protein family. m-Numb binds the post-CDC10/ankyrin repeat region of Notch (70). Chicken Numb protein binds the cytoplasmic region (C-terminal PEST region) of the Notch1 protein and blocks the nuclear transport of a truncated form of Notch1 (65). The findings from both groups suggested that the physical interaction between Numb and Notch1 may reduce the Notch1 activity (65, 70, 71). Correspondingly, we could demonstrate that m-Numb overexpression antagonized the dominant active Notch1-induced HES1 promoter transactivation (Fig. 6B). Furthermore, vertebrate Numb has been shown to play important roles in neural differentiation. Targeted disruption of the m-numb gene in mice results in severe defects in cortical neurogenesis (70). Overexpression of Numb protein promoted the differentiation of the MNS70 cell line of rat neural progenitor cells (37) (mouse Numb) (Tokunaga et al., unpublished results) and of chick neural progenitor cells (chick Numb) (65). Thus, it was expected that proper regulation of vertebrate Numb gene expression would be very important in the control of normal neural differentiation.

Msi1 inhibits m-numb expression at the translational level. We next examined the physiological role of the Msi1-numb mRNA interaction in regulating gene expression. Since Msi1 is predominantly and uniformly localized in the cytoplasm of immature neural cells (28, 52), not in their nuclei, Msi1 is not likely to participate in pre-mRNA splicing. The present results indicated that exogenously expressed Msi1 repressed the expression of endogenous m-Numb protein without altering its mRNA level (Fig. 5A and B). Msi1 also repressed the expression of a luciferase reporter containing the 3'-UTR of m-numb cDNA without affecting the reporter's steady-state RNA level (Fig. 5E). Furthermore, Msi1 comigrated with polysomes and monoribosomes and ribosome subunits through sucrose gradient centrifugation using lysate of NIH 3T3 cells infected with Adex-FLAGMsi (Fig. 5F) and localized to free or membrane-associated ribosomes in the cytoplasm of neural progenitor cells as viewed by electron microscopy (unpublished results). These subcellular localization studies on Msi1 suggest that Msi1 is likely to associate with ribosomes and regulate gene expression of downstream targets translationally. Taken together, these observations suggested that Msi1 is likely to repress the expression of the m-Numb protein at the level of translation rather than by reducing the RNA stability mediating the 3'-UTR of m-numb RNA.

HES1 promoter transactivation by Msi1 in nonneuronal cell line NIH 3T3 cells. NIH 3T3 fibroblast cells are widely used together with C2C12 cells to determine whether unknown factors, which are expressed in hematopoietic tissue or other tissues, can modulate Notch signaling (9, 41, 42, 50, 51, 67). One of the advantages of NIH 3T3 cells is the endogenous expressions of essential members of the Notch signaling pathway (Notch receptors, RBP-J, Deltex, m-Numb, etc.) at moderate levels. For example, RBP-J, which cotranslocates with the activated form of Notch1 and stimulates the transcriptional activity of HES1 promoter, is endogenously expressed in NIH 3T3 cells at a level high enough to activate the HES1 promoter upon the introduction of the dominant active form of Notch1. Thus, functions of a certain gene product can be investigated by assaying the level of HES1 promoter transactivation when it is coexpressed with the dominant active form of Notch1.

Biological significance of m-Numb down-regulation by Musashi1: self-renewing activity and survival of neural stem cells and possible involvement of Msi1 in diseases. What is the biological significance of the putative translational repression of the m-numb gene by Msi1? Previously, we demonstrated using neurospheres that mammalian Notch/HES1 signaling is essential for the self-renewing activity of neural stem cells and for the repression of their commitment to neuronal lineage (39). Furthermore, Notch signaling, which usually directs cells toward a nonneuronal fate, also has an antiapoptotic function in various cell types (3, 4). Consistent with the fact that Numb family proteins inhibit Notch signaling (14, 18, 65, 71), we and others have observed that overexpression of the vertebrate Numb protein in neural progenitor cells causes cell death (in the absence of caspase inhibitor) or neuronal differentiation (in the presence of caspase inhibitor) (65; Tokunaga et al., unpublished results). Thus, the translational repression of m-Numb expression by Msi1 could promote the self-renewing activity and/or survival of neural stem cells through the modulation of Notch signaling. To test the roles of Msi1 in the maintenance and differentiation of neural stem cells, loss-of-function studies are currently being performed.