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Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

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Scribble KD alters polyribosome distribution of Snail mRNA and increases translation of the Snail mRNA.A, Snail mRNA levels were not significantly changed in Scribble KD cells, as assessed by RT-PCR. B, Snail mRNA stability did not differ significantly between Scribble KD cells and control cells during up to 4 h of exposure to the transcription inhibitor actinomycin D (0.5 μg/ml). C, Snail protein synthesis rates were measured in a time course by proteasome inhibitor MG132 (20 μm) in Scribble KD and control cells. D, Snail protein degradation rates (indicated by slope of lines) did not differ significantly between Scribble KD and control cells following exposure to translation inhibitor cycloheximide (CHX, 0.5 μg/ml). E, relative polyribosome distribution of Snail mRNA in Scribble KD and control cells was analyzed by sucrose gradient centrifugation. mRNPs, messenger ribonucleoprotein particles. Unb, unbound RNPs, Fractions 1 and 2. Mono, monoRNPs (40S, 60S, 80S), fractions 3–5. Polysomes: polyribosomes, fractions 6–11. Representative data are from three independent experiments. F, analysis of RNP protein content in polyribosome fractions. Polyribosome distributions (12 fractions each) from control and Scribble KD cells in the CRL-1848 cell line were isolated as above, and RNA-binding protein content was analyzed by Western blotting; β-actin served as an internal control.
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Figure 3: Scribble KD alters polyribosome distribution of Snail mRNA and increases translation of the Snail mRNA.A, Snail mRNA levels were not significantly changed in Scribble KD cells, as assessed by RT-PCR. B, Snail mRNA stability did not differ significantly between Scribble KD cells and control cells during up to 4 h of exposure to the transcription inhibitor actinomycin D (0.5 μg/ml). C, Snail protein synthesis rates were measured in a time course by proteasome inhibitor MG132 (20 μm) in Scribble KD and control cells. D, Snail protein degradation rates (indicated by slope of lines) did not differ significantly between Scribble KD and control cells following exposure to translation inhibitor cycloheximide (CHX, 0.5 μg/ml). E, relative polyribosome distribution of Snail mRNA in Scribble KD and control cells was analyzed by sucrose gradient centrifugation. mRNPs, messenger ribonucleoprotein particles. Unb, unbound RNPs, Fractions 1 and 2. Mono, monoRNPs (40S, 60S, 80S), fractions 3–5. Polysomes: polyribosomes, fractions 6–11. Representative data are from three independent experiments. F, analysis of RNP protein content in polyribosome fractions. Polyribosome distributions (12 fractions each) from control and Scribble KD cells in the CRL-1848 cell line were isolated as above, and RNA-binding protein content was analyzed by Western blotting; β-actin served as an internal control.

Mentions: To analyze the molecular mechanism of the effect of Scribble on Snail accumulation, we examined Snail mRNA levels and Snail mRNA stability upon exposure to the transcription inhibitor actinomycin D. Neither the level nor stability of the transcript were significantly changed in response to Scribble KD compared with control cells (Fig. 3, A and B), suggesting that Scribble may regulate Snail accumulation at a post-transcriptional level. Following administration of MG132 at various time points, the rate of Snail protein synthesis was higher in Scribble KD cells while compared with control cells (Fig. 3C). The results from the time course of treatment in Scribble KD cells with cycloheximide, a protein synthesis inhibitor, indicated no apparent changes in the degradation or the half-life of Snail protein (Fig. 3D). We therefore supposed that Scribble regulates Snail accumulation at the level of translation. To further substantiate this impact of Scribble KD on Snail translation, we performed polyribosome profiling of cytoplasmic lysates from Scribble KD and control cells. In control cells, ∼25% of Snail mRNA co-sedimented with unbound ribonucleoprotein particles (RNPs) and mono-bound RNPs, and the remaining ∼75% of Snail mRNA co-sedimented with polyribosomes. In KD Scribble cells, approximately ∼90% of Snail mRNA co-sedimented with polyribosomes (Fig. 3E). These results indicated that the KD of Scribble increased the proportion of Snail mRNA in polyribosome fractions, yielding increased translation of Snail transcript.


Loss of Scribble Promotes Snail Translation through Translocation of HuR and Enhances Cancer Drug Resistance.

Zhou Y, Chang R, Ji W, Wang N, Qi M, Xu Y, Guo J, Zhan L - J. Biol. Chem. (2015)

Scribble KD alters polyribosome distribution of Snail mRNA and increases translation of the Snail mRNA.A, Snail mRNA levels were not significantly changed in Scribble KD cells, as assessed by RT-PCR. B, Snail mRNA stability did not differ significantly between Scribble KD cells and control cells during up to 4 h of exposure to the transcription inhibitor actinomycin D (0.5 μg/ml). C, Snail protein synthesis rates were measured in a time course by proteasome inhibitor MG132 (20 μm) in Scribble KD and control cells. D, Snail protein degradation rates (indicated by slope of lines) did not differ significantly between Scribble KD and control cells following exposure to translation inhibitor cycloheximide (CHX, 0.5 μg/ml). E, relative polyribosome distribution of Snail mRNA in Scribble KD and control cells was analyzed by sucrose gradient centrifugation. mRNPs, messenger ribonucleoprotein particles. Unb, unbound RNPs, Fractions 1 and 2. Mono, monoRNPs (40S, 60S, 80S), fractions 3–5. Polysomes: polyribosomes, fractions 6–11. Representative data are from three independent experiments. F, analysis of RNP protein content in polyribosome fractions. Polyribosome distributions (12 fractions each) from control and Scribble KD cells in the CRL-1848 cell line were isolated as above, and RNA-binding protein content was analyzed by Western blotting; β-actin served as an internal control.
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Figure 3: Scribble KD alters polyribosome distribution of Snail mRNA and increases translation of the Snail mRNA.A, Snail mRNA levels were not significantly changed in Scribble KD cells, as assessed by RT-PCR. B, Snail mRNA stability did not differ significantly between Scribble KD cells and control cells during up to 4 h of exposure to the transcription inhibitor actinomycin D (0.5 μg/ml). C, Snail protein synthesis rates were measured in a time course by proteasome inhibitor MG132 (20 μm) in Scribble KD and control cells. D, Snail protein degradation rates (indicated by slope of lines) did not differ significantly between Scribble KD and control cells following exposure to translation inhibitor cycloheximide (CHX, 0.5 μg/ml). E, relative polyribosome distribution of Snail mRNA in Scribble KD and control cells was analyzed by sucrose gradient centrifugation. mRNPs, messenger ribonucleoprotein particles. Unb, unbound RNPs, Fractions 1 and 2. Mono, monoRNPs (40S, 60S, 80S), fractions 3–5. Polysomes: polyribosomes, fractions 6–11. Representative data are from three independent experiments. F, analysis of RNP protein content in polyribosome fractions. Polyribosome distributions (12 fractions each) from control and Scribble KD cells in the CRL-1848 cell line were isolated as above, and RNA-binding protein content was analyzed by Western blotting; β-actin served as an internal control.
Mentions: To analyze the molecular mechanism of the effect of Scribble on Snail accumulation, we examined Snail mRNA levels and Snail mRNA stability upon exposure to the transcription inhibitor actinomycin D. Neither the level nor stability of the transcript were significantly changed in response to Scribble KD compared with control cells (Fig. 3, A and B), suggesting that Scribble may regulate Snail accumulation at a post-transcriptional level. Following administration of MG132 at various time points, the rate of Snail protein synthesis was higher in Scribble KD cells while compared with control cells (Fig. 3C). The results from the time course of treatment in Scribble KD cells with cycloheximide, a protein synthesis inhibitor, indicated no apparent changes in the degradation or the half-life of Snail protein (Fig. 3D). We therefore supposed that Scribble regulates Snail accumulation at the level of translation. To further substantiate this impact of Scribble KD on Snail translation, we performed polyribosome profiling of cytoplasmic lysates from Scribble KD and control cells. In control cells, ∼25% of Snail mRNA co-sedimented with unbound ribonucleoprotein particles (RNPs) and mono-bound RNPs, and the remaining ∼75% of Snail mRNA co-sedimented with polyribosomes. In KD Scribble cells, approximately ∼90% of Snail mRNA co-sedimented with polyribosomes (Fig. 3E). These results indicated that the KD of Scribble increased the proportion of Snail mRNA in polyribosome fractions, yielding increased translation of Snail transcript.

Bottom Line: Furthermore, we demonstrate HuR can recognize AU-rich elements of the Snail-encoding mRNA, thereby regulating Snail translation.Moreover, loss of Scribble-induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway.Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

View Article: PubMed Central - PubMed

Affiliation: From the Key Laboratory of Nutrition and Metabolism, Key Laboratory of Food Safety Research, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.

Show MeSH
Related in: MedlinePlus