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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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Rescue of Esrp1-depleted cells and differentiation potential of Esrp1-depleted ES cells.A. Sequence of Esrp1 cDNA showing ShRNA binding site (in bold and underlined) and the position of inserted mutations (red). The corresponding amino acid sequence is shown as well. B. Alkaline phosphatase (ALP) staining of Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) (n = 3). C. qRT-PCR analysis of FGFR2 IIIc/IIIb ratio in Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) showing a reduction in this ratio upon rescue. RQ is relative quantity (n = 3). D. qRT-PCR analysis of Esrp1, Oct4, Nanog and Sox2 mRNA in Esrp1-depleted ES cells transfected with pEm or Esrp1* expression vectors showing reduction in the expression of these genes upon rescue. RQ is relative quantity (n = 3).
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pone-0072300-g003: Rescue of Esrp1-depleted cells and differentiation potential of Esrp1-depleted ES cells.A. Sequence of Esrp1 cDNA showing ShRNA binding site (in bold and underlined) and the position of inserted mutations (red). The corresponding amino acid sequence is shown as well. B. Alkaline phosphatase (ALP) staining of Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) (n = 3). C. qRT-PCR analysis of FGFR2 IIIc/IIIb ratio in Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) showing a reduction in this ratio upon rescue. RQ is relative quantity (n = 3). D. qRT-PCR analysis of Esrp1, Oct4, Nanog and Sox2 mRNA in Esrp1-depleted ES cells transfected with pEm or Esrp1* expression vectors showing reduction in the expression of these genes upon rescue. RQ is relative quantity (n = 3).

Mentions: In order to exclude the possibility of off-target effects of the ShRNAs, rescue experiments were performed using a mutant Esrp1. The four mutations inserted in the ShRNA binding site in pIBX-C-FF-EmGFP-B-ESRP1-2A plasmid (Figure 3A) did not alter ESRP1 expression when compared to a plasmid expressing the wild-type ESRP1 (Figure S4A) [33]. Importantly, transient introduction of this mutant plasmid into Esrp1-depleted ES cells reverted the phenotype observed after Esrp1 depletion. In particular, compared to cells transfected with the empty vector only, less ALP- positive colonies formed from Esrp1-depleted ES cells transfected with the ShRNA-immune Esrp1 (Figures 3B). Restoration of Esrp1 expression also induced a decrease in FGFR2 IIIc/IIIb ratio and in core pluripotency genes expression (Figure 3C and D, respectively) to a level that was comparable to that of Scr ES cells. Similar results were obtained with ES cells in a different genetic background (v6.5) (Figure S4B) or after knockdown with a second ShRNA against Esrp1 (Figure S4C). Taken together, these results indicate that Esrp1 was acting on self-renewal by regulating the expression of pluripotency transcription factors.


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)

Rescue of Esrp1-depleted cells and differentiation potential of Esrp1-depleted ES cells.A. Sequence of Esrp1 cDNA showing ShRNA binding site (in bold and underlined) and the position of inserted mutations (red). The corresponding amino acid sequence is shown as well. B. Alkaline phosphatase (ALP) staining of Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) (n = 3). C. qRT-PCR analysis of FGFR2 IIIc/IIIb ratio in Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) showing a reduction in this ratio upon rescue. RQ is relative quantity (n = 3). D. qRT-PCR analysis of Esrp1, Oct4, Nanog and Sox2 mRNA in Esrp1-depleted ES cells transfected with pEm or Esrp1* expression vectors showing reduction in the expression of these genes upon rescue. RQ is relative quantity (n = 3).
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Related In: Results  -  Collection

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pone-0072300-g003: Rescue of Esrp1-depleted cells and differentiation potential of Esrp1-depleted ES cells.A. Sequence of Esrp1 cDNA showing ShRNA binding site (in bold and underlined) and the position of inserted mutations (red). The corresponding amino acid sequence is shown as well. B. Alkaline phosphatase (ALP) staining of Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) (n = 3). C. qRT-PCR analysis of FGFR2 IIIc/IIIb ratio in Scr and Esrp1-depleted ES cells transiently transduced either with empty vector (pEm) and with the ShRNA-immune Esrp1-GFP cDNA (Esrp1*) showing a reduction in this ratio upon rescue. RQ is relative quantity (n = 3). D. qRT-PCR analysis of Esrp1, Oct4, Nanog and Sox2 mRNA in Esrp1-depleted ES cells transfected with pEm or Esrp1* expression vectors showing reduction in the expression of these genes upon rescue. RQ is relative quantity (n = 3).
Mentions: In order to exclude the possibility of off-target effects of the ShRNAs, rescue experiments were performed using a mutant Esrp1. The four mutations inserted in the ShRNA binding site in pIBX-C-FF-EmGFP-B-ESRP1-2A plasmid (Figure 3A) did not alter ESRP1 expression when compared to a plasmid expressing the wild-type ESRP1 (Figure S4A) [33]. Importantly, transient introduction of this mutant plasmid into Esrp1-depleted ES cells reverted the phenotype observed after Esrp1 depletion. In particular, compared to cells transfected with the empty vector only, less ALP- positive colonies formed from Esrp1-depleted ES cells transfected with the ShRNA-immune Esrp1 (Figures 3B). Restoration of Esrp1 expression also induced a decrease in FGFR2 IIIc/IIIb ratio and in core pluripotency genes expression (Figure 3C and D, respectively) to a level that was comparable to that of Scr ES cells. Similar results were obtained with ES cells in a different genetic background (v6.5) (Figure S4B) or after knockdown with a second ShRNA against Esrp1 (Figure S4C). Taken together, these results indicate that Esrp1 was acting on self-renewal by regulating the expression of pluripotency transcription factors.

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