Figure 5. Msi1 inhibited the 80S... | Rockefeller University Press

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).$

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).

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