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The RNA binding protein ESRP1 fine-tunes the expression of pluripotency-related factors in mouse embryonic stem cells.

Fagoonee S, Bearzi C, Di Cunto F, Clohessy JG, Rizzi R, Reschke M, Tolosano E, Provero P, Pandolfi PP, Silengo L, Altruda F - PLoS ONE (2013)

Bottom Line: Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells.We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading.ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy.

ABSTRACT
In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.

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Polysomal loading of pluripotency-related mRNAs.A i. Polysome profiles of Scr and Esrp1-depleted ES cells obtained using an ISCO-fractionator. ii. qRT-PCR analysis shows the abundance of Oct4, Nanog, Sox2, c-Myc, Esrp1 and Lin28 mRNA in pooled polysomal fractions. 18s mRNA was equally immunoprecipitated in both cell types and average threshold cycle (Ct) is shown. RQ is relative quantity (n = 3). B. Representative Western blot analysis of ESRP1, RPS6 and RPL4 expression in the sucrose gradient fractions obtained from ES cells lysates under basal conditions.
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pone-0072300-g007: Polysomal loading of pluripotency-related mRNAs.A i. Polysome profiles of Scr and Esrp1-depleted ES cells obtained using an ISCO-fractionator. ii. qRT-PCR analysis shows the abundance of Oct4, Nanog, Sox2, c-Myc, Esrp1 and Lin28 mRNA in pooled polysomal fractions. 18s mRNA was equally immunoprecipitated in both cell types and average threshold cycle (Ct) is shown. RQ is relative quantity (n = 3). B. Representative Western blot analysis of ESRP1, RPS6 and RPL4 expression in the sucrose gradient fractions obtained from ES cells lysates under basal conditions.

Mentions: It is generally believed that mRNAs actively being translated are associated with polysomes and that an increased polysome association indicates an increase in translation efficiency [40]. Based on this assumption, to provide further evidence supporting the inhibitory role of Esrp1 on mRNA translation, we performed polysomal profiling [32]. Cytoplasmic extracts from Scr and Esrp1-depleted ES cells were subjected to sucrose gradient fractionation and total RNA was extracted from polysomal fractions. While there was no difference in the ISCO fractionator-generated polysome profile of Scr and Esrp1-depleted ES cells (Figure 7A), the amount of core pluripotency-related factors and c-Myc mRNAs in the pooled polysomal fractions was significantly higher upon depletion of Esrp1 compared to controls (Figure 7A). These results suggest that the increased protein levels of pluripotency factors shown in Figure 2D are at least partially due to their increased translation. On the other hand, Nanog and Lin28 mRNA loading did not significantly differ between Scr and Esrp1-depleted ES cells (Figure 7A). To further investigate in which sucrose gradient fractions ESRP1 was found in ES cells under basal conditions, we extracted proteins from the different fractions. The results show that ESRP1 is not located in the polysomal fractions (Figure 7B), suggesting that ESRP1 may sequester pluripotency-related mRNAs away from the polysomes. Taken together, our findings indicate that depletion of ESRP1 in ES cells resulted in alteration in the level of expression of core transcription factors and the resultant cumulative effect of these changes affects early differentiation of these cells.


The RNA binding protein ESRP1 fine-tunes the expression of pluripotency-related factors in mouse embryonic stem cells.

Fagoonee S, Bearzi C, Di Cunto F, Clohessy JG, Rizzi R, Reschke M, Tolosano E, Provero P, Pandolfi PP, Silengo L, Altruda F - PLoS ONE (2013)

Polysomal loading of pluripotency-related mRNAs.A i. Polysome profiles of Scr and Esrp1-depleted ES cells obtained using an ISCO-fractionator. ii. qRT-PCR analysis shows the abundance of Oct4, Nanog, Sox2, c-Myc, Esrp1 and Lin28 mRNA in pooled polysomal fractions. 18s mRNA was equally immunoprecipitated in both cell types and average threshold cycle (Ct) is shown. RQ is relative quantity (n = 3). B. Representative Western blot analysis of ESRP1, RPS6 and RPL4 expression in the sucrose gradient fractions obtained from ES cells lysates under basal conditions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755004&req=5

pone-0072300-g007: Polysomal loading of pluripotency-related mRNAs.A i. Polysome profiles of Scr and Esrp1-depleted ES cells obtained using an ISCO-fractionator. ii. qRT-PCR analysis shows the abundance of Oct4, Nanog, Sox2, c-Myc, Esrp1 and Lin28 mRNA in pooled polysomal fractions. 18s mRNA was equally immunoprecipitated in both cell types and average threshold cycle (Ct) is shown. RQ is relative quantity (n = 3). B. Representative Western blot analysis of ESRP1, RPS6 and RPL4 expression in the sucrose gradient fractions obtained from ES cells lysates under basal conditions.
Mentions: It is generally believed that mRNAs actively being translated are associated with polysomes and that an increased polysome association indicates an increase in translation efficiency [40]. Based on this assumption, to provide further evidence supporting the inhibitory role of Esrp1 on mRNA translation, we performed polysomal profiling [32]. Cytoplasmic extracts from Scr and Esrp1-depleted ES cells were subjected to sucrose gradient fractionation and total RNA was extracted from polysomal fractions. While there was no difference in the ISCO fractionator-generated polysome profile of Scr and Esrp1-depleted ES cells (Figure 7A), the amount of core pluripotency-related factors and c-Myc mRNAs in the pooled polysomal fractions was significantly higher upon depletion of Esrp1 compared to controls (Figure 7A). These results suggest that the increased protein levels of pluripotency factors shown in Figure 2D are at least partially due to their increased translation. On the other hand, Nanog and Lin28 mRNA loading did not significantly differ between Scr and Esrp1-depleted ES cells (Figure 7A). To further investigate in which sucrose gradient fractions ESRP1 was found in ES cells under basal conditions, we extracted proteins from the different fractions. The results show that ESRP1 is not located in the polysomal fractions (Figure 7B), suggesting that ESRP1 may sequester pluripotency-related mRNAs away from the polysomes. Taken together, our findings indicate that depletion of ESRP1 in ES cells resulted in alteration in the level of expression of core transcription factors and the resultant cumulative effect of these changes affects early differentiation of these cells.

Bottom Line: Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells.We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading.ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Turin, Italy.

ABSTRACT
In pluripotent stem cells, there is increasing evidence for crosstalk between post-transcriptional and transcriptional networks, offering multifold steps at which pluripotency can be controlled. In addition to well-studied transcription factors, chromatin modifiers and miRNAs, RNA-binding proteins are emerging as fundamental players in pluripotency regulation. Here, we report a new role for the RNA-binding protein ESRP1 in the control of pluripotency. Knockdown of Esrp1 in mouse embryonic stem cells induces, other than the well-documented epithelial to mesenchymal-like state, also an increase in expression of the core transcription factors Oct4, Nanog and Sox2, thereby enhancing self-renewal of these cells. Esrp1-depleted embryonic stem cells displayed impaired early differentiation in vitro and formed larger teratomas in vivo when compared to control embryonic stem cells. We also show that ESRP1 binds to Oct4 and Sox2 mRNAs and decreases their polysomal loading. ESRP1 thus acts as a physiological regulator of the finely-tuned balance between self-renewal and commitment to a restricted developmental fate. Importantly, both mouse and human epithelial stem cells highly express ESRP1, pinpointing the importance of this RNA-binding protein in stem cell biology.

Show MeSH
Related in: MedlinePlus