Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May 19;181(4):639-53.
doi: 10.1083/jcb.200708004.

Neural RNA-binding Protein Musashi1 Inhibits Translation Initiation by Competing With eIF4G for PABP

Affiliations
Free PMC article

Neural RNA-binding Protein Musashi1 Inhibits Translation Initiation by Competing With eIF4G for PABP

Hironori Kawahara et al. J Cell Biol. .
Free PMC article

Abstract

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural stem cells. We previously reported that Msi1 contributes to the maintenance of the immature state and self-renewal activity of neural stem cells through translational repression of m-Numb. However, its translation repression mechanism has remained unclear. Here, we identify poly(A) binding protein (PABP) as an Msi1-binding protein, and find Msi1 competes with eIF4G for PABP binding. This competition inhibits translation initiation of Msi1's target mRNA. Indeed, deletion of the PABP-interacting domain in Msi1 abolishes its function. We demonstrate that Msi1 inhibits the assembly of the 80S, but not the 48S, ribosome complex. Consistent with these conclusions, Msi1 colocalizes with PABP and is recruited into stress granules, which contain the stalled preinitiation complex. However, Msi1 with mutations in two RNA recognition motifs fails to accumulate into stress granules. These results provide insight into the mechanism by which sequence-specific translational repression occurs in stem cells through the control of translation initiation.

Figures

Figure 1.
Figure 1.
Identification of PABP as an Msi1-specific binding protein by the TAP method. (A) Msi1-bound proteins that were extracted from 293T cells expressing Flag-Msi1-TAP were resolved by SDS–PAGE, visualized by CBB staining (lanes 2 and 4), and compared with those of control Flag-TAP-expressing cells (lanes 1 and 3). The bound proteins in the TEV-digested extracts are shown in lanes 1 and 2; similarly, those of in the final extracts are shown in lanes 3 and 4. CBB-stained PABP, IMP, and Msi1 are indicated with arrowheads. (B) Msi1 colocalized with PABP and IMP3 in the cytoplasm. P19 cells were stained with anti-Msi1 (green) antibody, and anti-PABP (red, top) or anti-IMP3 (red, bottom) antibodies. Nuclei were stained with Hoechst (blue in P19 cells) in the merged image. (C) Immunoblottings after immunoprecipitation with ant-Msi1 antibody using E14 mouse brain extracts were performed with each antibody, respectively. (D) Protein extracts prepared from mouse brain at E16 were mixed with bacterially expressed and purified GST or GST-PABP fusion proteins. The GST fusion proteins were stained with CBB (lanes 1 and 2). Elutes were analyzed by immunoblotting using anti-eIF4G, anti-Msi1 14H1, or anti-eIF4E antibodies (lanes 3–6). (E) Double-immunohistochemistry of Msi1 (red) and PABP (green), eIF4G (green), or Sox1/(2)/3 (green) in coronal sections of the E14 forebrain. Sox1/(2)/3 is a marker for neural precursor cells. Inset in E shows a low magnification view of the main Msi1-expressing regions. CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone. Bars: 5 μm (B), 50 μm (E).
Figure 2.
Figure 2.
The C-terminal region of Msi1 that bound PABP is necessary for its function. (A) Illustration of proteins containing the T7-Msi1 variants: Msi1Amut (mutation in RRM1, and fails to bind mRNA: lane 2), Msi1Bmut (mutation in RRM2: lane 3), and a series of Msi1C-terminal deletions (lanes 4–11). (B) Immunoprecipitation using the T7-Msi1 variants was performed and various T7-Msi1 mutants bound to Myc-PABP (middle). The intensities of binding with PABP are illustrated to the right of panel A. (C) Illustration of the GST-Msi1 variants. (D) GST-Msi1 variants or GST as a control were coimmunoprecipitated with Myc-PABP in 293T cells using glutathione-Sepharose 4B (middle). PABP bound to Msi1 variants was immunoblotted using an anti-Myc antibody and is indicated (middle). (E) The in vitro–transcribed reporter mRNAs are illustrated at top (left, mRNA containing MCS; right, mRNA containing MCSmut), were translated in RRL with equimolar amounts of purified various GST proteins, and the luciferase activity was measured at each time point (0–90 min). The values represent mean ± SD; n = 5.
Figure 3.
Figure 3.
Msi1 competed with binding of eIF4G to PABP. (A) Illustration of the PABP variants. (B) Flag-Msi1 or Flag-eIF4GN-(1–582) was coimmunoprecipitated with Myc-PABP variants in 293T cells using anti-FLAG resin. Notably, Msi1 and eIF4G bound to a common domain within PABP (middle, bottom). (C) In vitro competition assay between purified GST-Msi1 and purified Flag-eIF4G (45–1560)-His immobilized FLAG resin. The CBB-stained, purified fusion proteins Flag-eIF4G (41–1560)-His, GST-PABP, GST, GST-Msi1-D2, and GST-Msi1 are shown (left panel, lanes 1–5). (D–F) Analysis of the kinetics of PABP's interaction with Msi1 or eIF4G by the QCM. (D) Illustration of the His-tagged proteins immobilized on the QCM plate and GST-PABP. The His-tag proteins were anchored to the QCM plate by an anti-His antibody. (E) Curves showing the time course of the changes in frequency for the proteins coated on the QCM plate, His-eIF4G 41-244mut, His-Msi1-D2, His-eIF4G 41–244, His-Msi1, and Flag-eIF4G (41–1560)-His, in response to the addition of 100 nM GST-PABP. (F) Summary of the kinetics parameters for the binding of PABP to Msi1 or eIF4G on the QCM; for a more detailed description see Materials and methods. (G) In vivo competition assay using 293T cells expressing Flag-eIF4GN and Myc-Msi1. The quantitative analysis was performed with Multigauge software (Fujifilm) in C and G (n = 5, mean ± SEM; *, P < 0.01 vs. control; †, P < 0.05 vs. control).
Figure 4.
Figure 4.
Cellular localization of Msi1. (A) Msi1 localizes to cytoplasmic foci. P19 cells treated with (right columns; 44°C for 30 min) or without (left columns) heat stress were stained with anti-Msi1 (green), and anti-hRAP55 (red, top) or anti-Dcp1a (red, bottom) antibodies, respectively. Nuclei were stained with TO-PRO-3 (blue) in the merged images. The white arrowheads and white arrow indicate PBs and SGs, respectively. Bars, 5 μm. (B) Msi1-positive granules were analyzed by two methods assessing the percent colocalization (1) or weighted colocalization coefficient (2) of their ratio to Dcp1a-, hRAP55-, PABP-, and eIF4G-containing granules. Msi1 mostly localized to SGs, but some was localized to PBs. (C) Association of Msi1 with heavy-sedimenting particles in an RNA-dependent manner. Each subcellular fraction with (lanes 4–6) or without (lanes 1–3) RNase A treatment after ultracentrifugation (top panels). S100, supernatants after ultracentrifugation; P100, pellets after ultracentrifugation. Total RNAs purified from S100 (lanes 2 and 5) and P100 (lanes 3 and 6) are shown (bottom panel). (D) P19 cells treated with (left) or without (right) heat stress were separated by 15–40% sucrose density gradients. Immunoblots of the gradient fractions were probed using antibodies against the indicated proteins (bottom panels).
Figure 5.
Figure 5.
Msi1 inhibited the 80S ribosome initiation complex formation on mRNA. (A) Illustration of the in vitro–transcribed reporter mRNAs (top). The reporter mRNA and purified GST-tagged proteins were incubated with RRL at 30°C for 90 min. Msi1 repressed the cap-dependent and IRES-dependent translation. The relative luciferase activity value represents the mean ± SD: (n = 4; *, P < 0.01 vs. buffer). (B) Cap column assay was performed in HeLa cells expressing Flag-GST, Flag-Msi1, or Flag-eIF4G-MD, which contains the eIF4E-binding domain. (C and D) Msi1 colocalized with translation initiation factors in P19 cells (C) and in cultured hippocampal neurons (D). Treatment with heat stress (44°C for 30 min) is indicated at the right (C) and bottom (D) of columns. Cells were stained with anti-Msi1 (green), anti-eIF4E, anti-eIF4G, and anti-PABP antibodies (red). Nuclei were stained with Hoechst (blue) in the merged images. Msi1 accumulates in SGs under heat stress. Bars, 5 μm. (E and F) 80S or 40S ribosome binding assay using in vitro–transcribed reporter mRNAs containing MCS-poly(A). Curves show the relative radioactivity of each fraction from reaction mixtures supplemented with equimolar amounts of GST (purple line), GST-Msi1 (green line), or GST-Msi1-D2 (red line), or buffer as a control (blue line). The percentage of the total recovered count was plotted against the fraction number (top panels). The RNAs purified from each fraction are shown (bottom panels). These results were reproduced in three independent experiments. (E) Peaks (fraction 19 or 20) corresponding labeled reporter mRNA in a complex with 80S ribosomes are indicated with arrows (top). The peak of 28S and 18S rRNA was found in fraction 19 or 20 (bottom). With GST-Msi1 addition, the 80S ribosome complex formation decreased to 52.7 ± 3.0% (n = 4; mean ± SD; P < 0.001) of the buffer control level. (F) Peaks (fraction 20 or 21) corresponding to labeled reporter mRNA in a complex with 48S ribosomes are indicated with arrows (top). The peak of 18S rRNA was found in fraction 20 or 21 (bottom).
Figure 6.
Figure 6.
Two RRMs of Msi1 as a regulated modifier domain of its cytoplasmic localization. Illustration of Msi1 variants that were modifications of the constructs described in Fig. 2 A. (B) HeLa cells were transfected with constructs expressing Flag-Msi1, Flag-Msi1ABmut, 3xFlag-Msi1-D2 (1–189 and 235–362), T7-Msi1, T7-Msi1Amut, T7-Msi1Bmut, T7-Msi1CdelG (1–234), and T7-Msi1CdelI (1–189). HeLa cells treated with (44°C for 30 min; left panels) or without (right panels) heat stress were stained with anti-Flag (green), anti-T7 (green), anti-eIF4G (red), and anti-Dcp1a (red, C) antibodies, respectively. Nuclei were stained with Hoechst (blue) in the merged images. The white arrowheads and white arrow indicate PBs and SGs, respectively. Bars, 5 μm.
Figure 7.
Figure 7.
A working model for targeted translational repression by Msi1. Msi1 interacts with the 3′ UTR of its target mRNA and PABP, and subsequently inhibits translation initiation by competing with eIF4G for PABP. These sequential events inhibit formation of the 80S ribosome complex.

Similar articles

See all similar articles

Cited by 81 articles

See all "Cited by" articles

References

    1. Anderson, P., and N. Kedersha. 2006. RNA granules. J. Cell Biol. 172:803–808. - PMC - PubMed
    1. Aoki, K., Y. Ishii, K. Matsumoto, and M. Tsujimoto. 2002. Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution. Nucleic Acids Res. 30:5182–5192. - PMC - PubMed
    1. Barbee, S.A., P.S. Estes, A.M. Cziko, J. Hillebrand, R.A. Luedeman, J.M. Coller, N. Johnson, I.C. Howlett, C. Geng, R. Ueda, et al. 2006. Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron. 52:997–1009. - PMC - PubMed
    1. Battelli, C., G.N. Nikopoulos, J.G. Mitchell, and J.M. Verdi. 2006. The RNA-binding protein Musashi-1 regulates neural development through the translational repression of p21WAF-1. Mol. Cell. Neurosci. 31:85–96. - PubMed
    1. Bhattacharyya, S.N., R. Habermacher, U. Martine, E.I. Closs, and W. Filipowicz. 2006. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell. 125:1111–1124. - PubMed

Publication types

MeSH terms

Feedback