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. 2015 Oct 1;29(19):2004-9.
doi: 10.1101/gad.267112.115.

Role of the small subunit processome in the maintenance of pluripotent stem cells

Kwon Tae You  1 , Joha Park  1 , V Narry Kim  1
Affiliations

Role of the small subunit processome in the maintenance of pluripotent stem cells

Kwon Tae You et al. Genes Dev. .

Abstract

RNA-binding proteins (RBPs) play integral roles in gene regulation, yet only a small fraction of RBPs has been studied in the context of stem cells. Here we applied an RNAi screen for RBPs in mouse embryonic stem cells (ESCs) and identified 16 RBPs involved in pluripotency maintenance. Interestingly, six identified RBPs, including Krr1 and Ddx47, are part of a complex called small subunit processome (SSUP) that mediates 18S rRNA biogenesis. The SSUP components are preferentially expressed in stem cells and enhance the global translational rate, which is critical to sustain the protein levels of labile pluripotency factors such as Nanog and Esrrb. Furthermore, the SSUP proteins are required for efficient reprogramming of induced pluripotent stem cells. Our study uncovers the role of the SSUP and the importance of translational control in stem cell fate decision.

Keywords: RNA-binding protein; SSU processome; embryonic stem cell; pluripotency; ribosome; translational control.

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Figures

Figure 1.
Figure 1.
RNAi screen identifying novel RBPs involved in the regulation of pluripotency. (A) Relative expression of Nanog, Cdx2, and Fgf5 mRNA after knockdown of 16 validated RBPs is shown. The SSUP genes are in red letters. All of the error bars represent the mean ± SD of triplicates or two experiments (when indicated). All of the mRNA levels were normalized against Actb mRNA or others (when indicated). (B) A box plot showing reduced expression of Nanog mRNA upon knockdown of SSUP genes. (C) Western blotting shows the relative expression of Nanog and Pou5f1 after depletion of SSUP genes for 3 d. Relative values were normalized to Gapdh levels. (*) P < 1.0 × 10−7 by two-tailed t-test. See also Supplemental Figure S1.
Figure 2.
Figure 2.
The SSUP is required for the maintenance of ESC identity. (A) Immunostaining shows the expression of Nanog and Krr1 in ESCs transfected with siKrr1 (stained 3 d following knockdown). Bars, 25 μm. (B) Overexpression of krr1 rescues the reduction of Nanog in Krr1-depleted cells. (C) Differentially expressed gene (DEG) analysis of RNA-seq reveals the DEGs after 2 d of Krr1 knockdown (1% false discovery rate [FDR]). (D) Down-regulation of SSUP factors reduces the reprogramming efficiency. Lentiviruses harboring each shRNA were cotransduced with lentivirus containing OSKM (Oct4, Sox2, Klf4, and c-Myc) reprogramming factors. The alkaline phosphatase (AP)-positive colonies were counted 11 d after transduction. (*) P < 0.05; (**) P < 0.005 by two-tailed t-test. See also Supplemental Figures S2–S4.
Figure 3.
Figure 3.
The SSUP is preferentially expressed in pluripotent stem cells. (A) Western blotting determines the levels of SSUP components during EB differentiation. (‡) Nonspecific band. (B) Box plot presenting the change of mRNAs during EB differentiation compared with ESCs (Liu et al. 2011). (*) P < 1.0 × 10−5 by two-tailed t-test. See also Supplemental Figures S5 and S6.
Figure 4.
Figure 4.
The SSUP contributes to pluripotency by enhancing general translational capacity. (A) Polysome profiles showing the changes in the ribosome pool upon Krr1 knockdown. (B) Metabolic labeling determines the bulk protein synthesis rate in control (siNC) and Krr1-depleted (siKrr1) cells. (C) Metabolic labeling reveals the differences in translational activity of ESCs and differentiating progenies (EBs and retinoic acid [RA]-treated cells). (D) Polysome profiles showing translational repression in RA-treated differentiating cells. (E) Schematic diagram of ribosome profiling, which can measure the translational efficiency (TE) of individual genes. (F) A scatter plot showing the correlation of TE between controls and knockdown samples. The R-value refers to Pearson correlation coefficient. (*) P < 0.05 by two-tailed t-test. See also Supplemental Figures S7 and S9–S11.
Figure 5.
Figure 5.
Regulation of global protein synthesis influences ESC identity. (A) Translational repression affects the level of several core ESC TFs. Ncl and Ubf were used as control nuclear proteins. Quantitation of protein levels by Western blotting and of mRNA levels by qRT–PCR is shown at the right. (B) Metabolic labeling indicates the changes in protein synthesis rate after treatment with 50 μM 4EGI-1 for 3 d. (C) Treatment of 4EGI-1 as in B affects the expression of pluripotent genes. (D) The same number of 4EGI-1-treated cells was reseeded, and, after 3 d of incubation in ESC culture conditions, the AP-positive colonies were counted. (E) Model depicting the mechanism of SSUP regulation and stem cell control. (*) P < 1.0 × 10−7 by two-tailed t-test.
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References

    1. Chia NY, Chan YS, Feng B, Lu X, Orlov YL, Moreau D, Kumar P, Yang L, Jiang J, Lau MS, et al. 2010. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468: 316–320. - PubMed
    1. Deisenroth C, Zhang Y. 2010. Ribosome biogenesis surveillance: probing the ribosomal protein–Mdm2–p53 pathway. Oncogene 29: 4253–4260. - PubMed
    1. Ding L, Paszkowski-Rogacz M, Nitzsche A, Slabicki MM, Heninger AK, de Vries I, Kittler R, Junqueira M, Shevchenko A, Schulz H, et al. 2009. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell 4: 403–415. - PubMed
    1. Ding J, Xu H, Faiola F, Ma'ayan A, Wang J. 2012. Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res 22: 155–167. - PMC - PubMed
    1. Evans MJ, Kaufman MH. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156. - PubMed

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