Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells

Abstract

Lin28 is highly expressed in human and mouse embryonic stem (ES) cells. Here, we show that in mouse ES cells, specific repression of Lin28 results in decreased cell proliferation, while overexpression of Lin28 accelerates cell proliferation. Further, Lin28 associates specifically with ribonucleoprotein particles containing mRNAs for cyclins A and B and cdk4. Importantly, changes in Lin28 levels lead to corresponding changes in the levels of these proteins, and sequences from the 3' untranslated regions of cyclin B and cdk4 mRNAs exhibit stimulatory effects on translation of reporter genes in a Lin28-dependent fashion. Thus, we postulate that Lin28 may play a role in the regulation of translation of genes important for the growth and maintenance of pluripotent cells.

FIGURE 1.

Lin28 expression alters cell cycle structure. (A,B) ES cells were transfected with the indicated siRNAs or plasmid DNAs as previously described (Zhang et al. 2007), respectively. Forty-eight hours later, cells were pulse-labeled with BrdU for 1 h and processed for flow cytometric analysis. The percentages of cells in the G₁-, S-, or G₂/M phase are shown, together with representative dot plots. Values are the mean of two experiments.

FIGURE 2.

Lin28 associates with a specific subset of mRNAs. (A) Flag-Lin28 was transfected into mouse ES cells. Immunoprecipitation (IP) was carried out 24 h later and RNP-associated RNAs analyzed by RT-qPCR. mRNA levels present in the anti-Flag relative to control IgG complexes are shown. Each bar represents mean ± SD (n = 3). (B) Steady-state mRNA levels plotted relative to those of β-actin mRNA. Each bar represents mean ± SD (n = 3).

FIGURE 3.

Lin28 may affect the translation of its associated mRNAs. (A) Mouse ES cells were transfected with Lin28 siRNA or control siRNA, and cell proteins isolated and subjected to Western blot analysis. (Top) Western blots using antibodies. (Bottom) Western blots of the same membranes but using an antibody specific for gapdh. Numbers in italics at the bottom indicate protein levels as percentage of Lin28 siRNA treated compared with control siRNA treated after normalization using gapdh signals. (B) Luciferase reporter constructs (Vasudevan and Steitz 2007) containing sequences from cdk4 3′ UTR (UTR), the middle (B1U2), or first (B1U1) part of cyclin B 3′ UTR, were transfected into HEK293 cells together with increasing amounts of Flag-Lin28. Luciferase activities were measured 24 h after the transfection and relative luciferase activities plotted. Luciferase activities from cells without Flag-Lin28 cotransfection were arbitrarily set as 100%. Results are representatives of at least three independent experiments. Numbers shown are means of duplicate samples. Shown on the right are schematic mappings of the individual fragments. Figures are not drawn to scale.