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Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation.

Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R - Nat. Genet. (2008)

Bottom Line: These miRNAs function by suppressing several key regulators of the G1-S transition.These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells.Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

View Article: PubMed Central - PubMed

Affiliation: Institute for Regeneration Medicine, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

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Screening for miRNAs that rescue the proliferation defects of Dgcr8 Δ/Δ ES cells. (a) Screening strategy. Proliferation of ES cells transfected with individual miRNA mimics was first evaluated by the MTT assay. The positive hits were then assessed for their ability to rescue the G1 accumulation defects of Dgcr8 Δ/Δ ES cells. (b) Z-scores for individual miRNA mimics. Shown are average Z-scores from triplicates. Error bar indicates the range of triplicates. (c) Top 14 positive hits with Z-score > 3 (P value < 0.001). The growth rate was normalized to mock transfected DGCR8 Δ/Δ ES cells. (d) 11 positive hits share similar seed sequence. Seed sequences are highlighted in gray box.
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Figure 1: Screening for miRNAs that rescue the proliferation defects of Dgcr8 Δ/Δ ES cells. (a) Screening strategy. Proliferation of ES cells transfected with individual miRNA mimics was first evaluated by the MTT assay. The positive hits were then assessed for their ability to rescue the G1 accumulation defects of Dgcr8 Δ/Δ ES cells. (b) Z-scores for individual miRNA mimics. Shown are average Z-scores from triplicates. Error bar indicates the range of triplicates. (c) Top 14 positive hits with Z-score > 3 (P value < 0.001). The growth rate was normalized to mock transfected DGCR8 Δ/Δ ES cells. (d) 11 positive hits share similar seed sequence. Seed sequences are highlighted in gray box.

Mentions: ES cells are derived from the inner cell mass of the blastocyst and have been used successfully as a tool to understand molecular mechanisms of early mammalian development2. Because ES cells can undergo infinite and rapid self-renewal without compromising pluripotency, they hold great potential for regenerative medicine. However, rapid proliferation could be detrimental if it leads to uncontrolled cell growth following transplantation into the host. The rapid proliferation of ES cells is thought to be due to their unique cell cycle structure, especially their shortened G1 phase3,4. Insights into the cell cycle control of ES cells have been gained by investigating the expression of cell cycle proteins5-8. Furthermore, small RNAs have been implicated in ES cell proliferation based on the phenotype of Dicer1 knockout ES cells9,10. More recently, using a Dgcr8 knockout model, we reported a proliferation defect in ES cells specifically deficient in a subclass of small RNAs, the canonical miRNAs1. These miRNA-deficient cells showed a relative increase in the number of cells in the G1 phase of the cell cycle. This finding suggests that miRNAs normally suppress inhibitors of the G1/S transition allowing the rapid transition from mitosis to S phase. However, confirmation of this hypothesis requires the identification of the specific miRNAs and targets involved in the process. Identification of individual miRNA function is complicated by the fact that miRNAs often function redundantly and exist at saturating levels in a wild-type background. To overcome this difficulty, we designed a screening strategy where individual miRNA mimics were reintroduced into an otherwise miRNA deficient background (Dgcr8 knockout ES cells) and then evaluated for rescue of the proliferation and cell cycle defects (Fig. 1a).


Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation.

Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R - Nat. Genet. (2008)

Screening for miRNAs that rescue the proliferation defects of Dgcr8 Δ/Δ ES cells. (a) Screening strategy. Proliferation of ES cells transfected with individual miRNA mimics was first evaluated by the MTT assay. The positive hits were then assessed for their ability to rescue the G1 accumulation defects of Dgcr8 Δ/Δ ES cells. (b) Z-scores for individual miRNA mimics. Shown are average Z-scores from triplicates. Error bar indicates the range of triplicates. (c) Top 14 positive hits with Z-score > 3 (P value < 0.001). The growth rate was normalized to mock transfected DGCR8 Δ/Δ ES cells. (d) 11 positive hits share similar seed sequence. Seed sequences are highlighted in gray box.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2630798&req=5

Figure 1: Screening for miRNAs that rescue the proliferation defects of Dgcr8 Δ/Δ ES cells. (a) Screening strategy. Proliferation of ES cells transfected with individual miRNA mimics was first evaluated by the MTT assay. The positive hits were then assessed for their ability to rescue the G1 accumulation defects of Dgcr8 Δ/Δ ES cells. (b) Z-scores for individual miRNA mimics. Shown are average Z-scores from triplicates. Error bar indicates the range of triplicates. (c) Top 14 positive hits with Z-score > 3 (P value < 0.001). The growth rate was normalized to mock transfected DGCR8 Δ/Δ ES cells. (d) 11 positive hits share similar seed sequence. Seed sequences are highlighted in gray box.
Mentions: ES cells are derived from the inner cell mass of the blastocyst and have been used successfully as a tool to understand molecular mechanisms of early mammalian development2. Because ES cells can undergo infinite and rapid self-renewal without compromising pluripotency, they hold great potential for regenerative medicine. However, rapid proliferation could be detrimental if it leads to uncontrolled cell growth following transplantation into the host. The rapid proliferation of ES cells is thought to be due to their unique cell cycle structure, especially their shortened G1 phase3,4. Insights into the cell cycle control of ES cells have been gained by investigating the expression of cell cycle proteins5-8. Furthermore, small RNAs have been implicated in ES cell proliferation based on the phenotype of Dicer1 knockout ES cells9,10. More recently, using a Dgcr8 knockout model, we reported a proliferation defect in ES cells specifically deficient in a subclass of small RNAs, the canonical miRNAs1. These miRNA-deficient cells showed a relative increase in the number of cells in the G1 phase of the cell cycle. This finding suggests that miRNAs normally suppress inhibitors of the G1/S transition allowing the rapid transition from mitosis to S phase. However, confirmation of this hypothesis requires the identification of the specific miRNAs and targets involved in the process. Identification of individual miRNA function is complicated by the fact that miRNAs often function redundantly and exist at saturating levels in a wild-type background. To overcome this difficulty, we designed a screening strategy where individual miRNA mimics were reintroduced into an otherwise miRNA deficient background (Dgcr8 knockout ES cells) and then evaluated for rescue of the proliferation and cell cycle defects (Fig. 1a).

Bottom Line: These miRNAs function by suppressing several key regulators of the G1-S transition.These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells.Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

View Article: PubMed Central - PubMed

Affiliation: Institute for Regeneration Medicine, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1-S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1-S transition of the ES cell cycle, enabling rapid proliferation of these cells. Our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes, as it overcomes the common problem of redundancy and saturation in the miRNA system.

Show MeSH
Related in: MedlinePlus