Histone H2a mRNA interacts with Lin28 and contains a Lin28-dependent posttranscriptional regulatory element

Abstract

Lin28 has been shown to block the processing of let-7 microRNAs implicated in the regulation of cell growth and differentiation. Here, we show that Lin28 also specifically associates with ribonucleoprotein particles containing the replication-dependent histone H2a mRNA in mouse embryonic stem cells. We further show that the coding region of H2a mRNA harbors high affinity binding sequences for Lin28 and that these sequences stimulate the expression of reporter genes in a Lin28-dependent manner. We suggest that a key function of Lin28 in the maintenance of pluripotency is to promote the expression of the H2a gene (and perhaps also other replication-dependent histone genes) at the posttranscriptional level in order to coordinate histone production with the unique proliferative properties of embryonic stem cells.

Figure 1.

Lin28 promotes cell proliferation. (a) ES cells were transfected with Lin28 siRNA, mock transfected, or transfected with control siRNA. Total cellular RNAs were extracted 24 h later, and the indicated mRNA levels determined by reverse transcription and quantitative real-time PCR (RT-qPCR). (b) ES cells were transfected with control siRNA (lane 1), Lin28 siRNA (lane 2), empty vector (lane 3) or Flag-Lin28 (lane 4). Cellular proteins were extracted 48 h (lanes 1 and 2) or 24 h (lanes 3 and 4) later, and protein levels determined by Western blot. Upper panel, Western blot using a Lin28-specific antibody. The band marked by an asterisk in lane 4 indicates Flag-Lin28, while bands below in lanes 1–4 indicate endogenous Lin28. Bottom panel, Western blot of the same membrane using an antibody specific for mouse beta-actin. (c and d) ES cells were transfected with control siRNA, Lin28 siRNA, empty vector, or Flag-Lin28, and cell numbers counted 48 h (c) or 24 h (d) following transfection. (e) Caspase-3/7 activities of ES cells were measured 24 and 48 h following the various transfections. Each bar represents mean ± SD (n = 3–4).

Figure 2.

Lin28 associates with a specific subset of mRNAs in mouse ES cells. (a) ES cells were transfected with Flag-Lin28 and RNPs isolated using anti-Flag. (b) RNPs were isolated from untransfected ES cells using anti-Lin28 antibody. (c) Relative mRNA levels after normalization against gapdh mRNA levels in the cell extract.

Figure 3.

XL and competition assays. Flag-Lin28 was transfected into HEK293 cells and cell extract prepared. (a) XL were carried out using radioactively labeled H2a coding region RNA in the absence (lane 2) or presence of increasing amounts of unlabeled H4 (lanes 3–5), H3 (lanes 6–8), H2b (lanes 9–11), B1U1 (lanes 12–14) or H2a (lanes 15–17) RNA, followed by IP. Total crosslinked product (5%) and immunoprecipitates were resolved by SDS–PAGE, followed by autoradiography. The bands marked with asterisk are Flag-Lin28. (b) Amounts of crosslinked Flag-Lin28 plotted against unlabeled competitor RNA in molar excess. Each point represents three independent experiments. Numbers are mean ± SD. (c) XL of radioactively labeled B1U1 RNA in the presence of increasing amounts of unlabeled B1U1 (lanes 3–5) or H2a (lanes 6–8) RNA.

Figure 4.

Luciferase activity assays. The indicated reporter constructs were transfected into NIH/3T3 cells that do not express endogenous Lin28, together with increasing amounts of Flag-Lin28. In addition, a Renilla reporter was included in all transfections for normalization purposes. Luciferase activities and mRNA levels were measured 24 h after transfection. Firefly luciferase activities [after normalization against Renilla luciferase (a and c)] and firefly mRNA levels (b) from cells without Flag-Lin28 transfection were arbitrarily set as 1. Numbers are mean ± SD (n = 3). F3-1X and F3-2X, firefly luciferase reporter containing one and two tandem copies of F3, respectively, inserted at its 3′UTR.

Figure 5.

A feedback model for the regulation of cell proliferation by Lin28 and let-7. Lin28 enhances the expression of genes that control proliferation, while let-7 inhibits them. We cannot exclude that at least some Lin28 targets are also regulated by let-7.