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First published online March 22, 2006
doi: 10.1242/10.1242/jcs.02852

Journal of Cell Science 119, 1442-1452 (2006)
Published by The Company of Biologists 2006
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A role for the ELAV RNA-binding proteins in neural stem cells: stabilization of Msi1 mRNA

Antonia Ratti1,*, Claudia Fallini1, Lidia Cova1, Roberto Fantozzi1, Cinzia Calzarossa1, Eleonora Zennaro1, Alessia Pascale2, Alessandro Quattrone3 and Vincenzo Silani1

1 Department of Neuroscience, `Dino Ferrari' Centre, University of Milan-IRCCS Istituto Auxologico Italiano, Via Zucchi 18, 20095 Cusano Milanino, Italy
2 Department of Experimental and Applied Pharmacology, University of Pavia, Via Taramelli 14, 27100 Pavia, Italy
3 Laboratory of Metabolomics and Systems Biology, Magnetic Resonance Center and FiorGen Foundation, University of Florence, Via Sacconi 6, 50019 Sesto Fiorentino, Italy


Figure 1
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  		Fig. 1. Computational analysis of the Msi1 3'UTR sequence and identification of the associated RBP activities. (A) Alignment of the mammalian Msi1 3'UTR sequences is shown: Homo sapiens (Hs, NM_002442) at the top, Rattus norvegicus (Rn, CD568097) in the middle and Mus musculus (Mm, NM_008629) at the bottom. Conserved nucleotides are highlighted in grey, whereas the ARE is indicated by the box. (B) The most stable RNA secondary structure of the mouse Msi1 3'UTR as predicted by the Sfold software (www.bioinfo.rpi.edu). The magnification shows the loop exposing the ARE sequence, indicated by the arrow. (C) UV crosslinking assay of Msi1 and Gap43 3'UTR riboprobes and lysates from whole mouse brain and NSCs. The 95 kDa complex is detected only when brain extracts were used. The 37 and 32 kDa RNA-binding activities specific for Msi1 sequence are indicated by asterisks. (D) Immunoprecipitation of Msi1 and Gap43 riboprobes with the anti-nELAV antibody after UV irradiation on brain and NSC extracts. A 42 kDa complex (arrowhead) was precipitated in all samples. (E) Immunoprecipitation of the NSC UV crosslinked mRNPs with the anti-nELAV and the anti-Msi1 antibodies, respectively.

 

Figure 2
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  		Fig. 2. nELAV RBPs are expressed in neurospheres and neural stem/progenitor cells in vitro. (A,B) The nELAV antibody (green) was used in combination with anti-Msi1 antibody (red) to label adult mouse neurospheres showing a strong overlapping expression of the two RBPs in the whole neurosphere (C, merged). (D,E) Colocalization of nELAV (green) and Msi1 (red) RBPs is more evident in single stem/progenitor cells. Most of the neurosphere-derived precursors show the expression of the two antigens with a different cellular distribution, mainly nuclear for Msi1 (F, merged, arrow). The same field is represented in the inset at a reduced size where nuclei are evidenced by DAPI counterstaining (arrow). (G,H,I) The proliferative state of the cell culture is attested by nELAV (green) and Ki67 (red) positive labeling. These two proteins are coexpressed in certain precursors, whereas nELAV is selectively expressed in others (arrowheads). The nuclear localization of Ki67 antigen is clearly visible (asterisks). Bars, 100 µm (A-C); 25 µm (D-I).

 

Figure 3
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  		Fig. 3. nELAV proteins are expressed in the proliferating cells of the adult rat SVZ. (A,B) Immunolabeling with anti-nELAV (red) and anti-Ki67 (green) antibodies revealed coexpression of the two markers (C, merged, higher magnification of the field indicated in B) in the sub-ependymal layer of the adult SVZ, where proliferating stem/progenitor cells are present. (D) A magnified image of the region indicated in C (bracket) clearly shows the distinct nuclear and cytoplasmic distribution of the Ki67 and nELAV markers, respectively, in the same cells. (E,F) nELAV (red) and Msi1 (green) RBPs colocalized in the SVZ (G, merged, higher magnification image of the field in F) and presented the same cytoplasmic distribution pattern (H, magnified picture of the region indicated in G by the bracket). Cc, corpus callosus; ChP, choroid plexus; CPu, caudate putamen; Lv, lateral ventricle; RMS, rostral migratory stream. Bars, 100 µm (A,B,E-F); 50 µm (C,G); 25 µm (D,H).

 

Figure 4
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  		Fig. 4. Real-time PCR-based identification and quantification of mRNAs associated to nELAV-mRNP complexes in adult neurospheres. Endogenous mRNPs were immunoprecipitated by anti-nELAV, anti-His5 and no antibody and the collected mRNA species were quantified by real-time RT-PCR. The normalized fold difference is indicated with respect to the sample where no antibody was used (mean value ± s.e.m.; one-way ANOVA, n=3, ***P<0.001 for anti-nELAV antibody compared with both controls).

 

Figure 5
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  		Fig. 5. HuD protein specifically recognizes and binds the Msi1 3'UTR sequence. (A) Schematic representation of the mouse Msi1 3'UTR (399 bp). ARE+ (230 bp) and ARE- (148 bp) deletion segments were obtained by PCR amplification with the indicated primer pairs (see Materials and Methods). (B) Left panel, polyacrylamide gel of UV-crosslinking assays on neurosphere extracts with the ARE+ and ARE- deletion segments. The binding activity was completely abolished in the absence of the ARE element. Msi1 full-length 3'UTR was used as a positive control (middle lane). Right panel, UV crosslinking assay of the recombinant HuD protein with the Msi1 3'UTR riboprobe compared with Gap43 as a control. The 42 kDa complex (arrowhead) can be detected. HuD specifically bound the deletion fragment ARE+, but not the ARE- sequence. (C) Left panel, competition experiments with the recombinant HuD protein were performed with the addition of a 100x molar excess of cold ARE+ and ARE- riboprobes. The Msi1-HuD complex is shown in the first lane before the addition of the cold competitors. Right panel, an excess of cold full-length Msi1 3'UTR completely inhibited the formation of the mRNP complex. The antisense Msi1 sequence (As) was not able to specifically bind HuD protein. (D) Dose-dependent binding-activity of the HuD protein (0.1, 0.2, 0.4, 1 µg) to the Msi1 3'UTR sequence.

 

Figure 6
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  		Fig. 6. The conserved ARE sequence affects Msi1 mRNA stability in vitro. (A) Decay assay of µmsi1ARE+ and µmsi1AS radio-labeled transcripts at different time points. The A60 labels represent the initial µmsi1ARE+ and µmsi1AS transcripts with 60 adenines in the polyA tail, whereas the A0 labels represent the corresponding deadenylated transcripts. (B) The deadenylation rate of the µmsi1ARE+ RNA changed after the addition of 0.75 µg of the HuD protein, with a decay pattern similar to that of the µmsi1AS transcript. The deadenylation/degradation profiles were highly reproducible (compare the µmsi1ARE+ decay in A and B).

 

Figure 7
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  		Fig. 7. nELAV-dependent upregulation of Msi1 protein after phorbol ester treatment. (A) After stimulation for 15 minutes with 100 nM PMA or DMSO, cell fractions (cytosol, membranes and cytoskeleton) were separated from human SH-SY5Y cells. Samples were analysed by western blotting with the indicated antibodies. (B) Densitometric analyses of western blot data normalized to {alpha}-tubulin protein content (mean ± s.d.; n=3, *P<0.05, ***P<0.001, Student's t-test).

 

Figure 8
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  		Fig. 8. Model of the hypothetical role of the nELAV RBPs during neurogenesis. In the temporal sequence of events that lead a proliferating NSC to become a differentiated neuron, Msi1 and nELAV RNA-binding activities are complementary and exert a different function on their target mRNAs. nELAV stabilization of the Msi1 transcript may prolong its expression during the gradual passage from proliferation to differentiation.

 

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© The Company of Biologists Ltd 2006