Transcripts that associate with the RNA binding protein, DEAD-END (DND1), in embryonic stem (ES) cells

Abstract

Background: The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates. It is also a testicular germ cell tumor susceptibility factor in mice. DND1 has been shown to interact with the 3'-untranslated region (3'-UTR) of mRNAs such as P27 and LATS2. Binding of DND1 to the 3'-UTRs of these transcripts blocks the inhibitory function of microRNAs (miRNA) from these transcripts and in this way DND1 helps maintain P27 and LATS2 protein expression. We found that DND1 is also expressed in embryonic stem (ES) cells. Because ES cells share similar gene expression patterns as germ cells, we utilized ES cells to identify additional candidate mRNAs that associate with DND1.

Results: ES cells are readily amenable to genetic modification and easier to culture in vitro compared to germ cells. Therefore, for the purpose of our study, we made a genetically modified, stable, human embryonic stem (hES) cell line that expresses hemagluttinin (HA)-tagged DND1 in a doxycycline (dox) regulatable manner. This line expresses modest levels of HA-DND1 and serves as a good system to study DND1 function in vitro. We used this stable cell line to identify the transcripts that physically interact with DND1. By performing ribonucleoprotein immunoprecipitation (RIP) followed by RT-PCR, we identified that transcripts encoding pluripotency factors (OCT4, SOX2, NANOG, LIN28), cell cycle regulators (TP53, LATS2) and apoptotic factors (BCLX, BAX) are specifically associated with the HA-DND1 ribonucleoprotein complex. Surprisingly, in many cases, bioinformatics analysis of the pulled-down transcripts did not reveal the presence of known DND1 interacting motifs.

Conclusions: Our results indicate that the inducible ES cell line system serves as a suitable in vitro system to identify the mRNA targets of DND1. The RIP-RT results hint at the broad spectrum of mRNA targets that interact with DND1 in ES cells. Based on what is known about DND1 function, our results suggest that DND1 may impose another level of translational regulation to modulate expression of critical factors in ES cells.

Figure 1

DND1 expression in hES cells. (a) Immunoblotting using anti-DND1 antibody of MEF, hES line H9, mouse testes (T) and spleen (S) (10 μg protein/lane). Control (C) is 293T cells transfected with HA-DND1 encoding expression vector. (bottom) Blot reprobed with anti-GAPDH. (b) RT-PCR to amplify DND1 in human testes (T), hES cells H1, H9, and MEFs. (+) lanes had superscript; (-) had no superscript during RT. (bottom) RT-PCR for HPRT. (c) . The first virus expresses rtTA that binds dox. The second virus carries dox-responsive promoter (sgTRE) that drives HA-DND1 expression. Dox also induces GFP expression, which allows identification of cells carrying both constructs. att (sites for recombinational cloning); LTR and ΔLTR (long terminal repeats); IRES (internal ribosome entry site). (d) FACS analysis indicates hES/HA-DND1 cells maintain high expression (77% +ve) of OCT4 and SSEA-4 (quadrant Q2, top right). (e) hES/HA-DND1 cells induced to express HA-DND1 upon dox treatment (100 to 1000 ng/mL) for 24 h. Immunoblotting using anti-HA antibody (top panel), anti-DND1 antibody (middle panel) and anti-GAPDH antibody (bottom panel) (20 μg hES/HA-DND1 lysates/lane). Relative HA-DND1 levels (fold HA) range from 1.6 - 3.7 fold with increasing dox. (Relative HA-DND1 of each lane is ratio of HA-DND1/GAPDH). The level of endogenous DND1 (DND1/GAPDH) in hES/HA-DND1 cells treated with 0 to 1000 ng/mL dox was 1.2, 0.9, 1.1, 1.1 and 0.7, respectively. The level of induced HA-DND1 to endogenous DND1 levels (HA/endog) was estimated to be 1.7 to 5.4-fold, with increasing dox.

Figure 2

The RIP-RT procedure. (a) Outline of RIP-RT procedure. hES/HA-DND1 cells were treated with and without dox. Anti-HA antibody was used to pull-down HA-DND1 together with associated mRNA. The mRNA was eluted and RT-PCR used to amplify and detect P27. (b) Details of the RIP procedure as described in [29]. hES/HA-DND1 cells were used to prepare lysates (mRNP lysates). A fraction of the lysate was used as input (I) for immunoprecipitation (IP) using anti-HA antibody linked to beads. After an overnight incubation, the input was centrifuged. The supernatant (S) was removed. RNA was extracted from the beads (RIP fraction), I and S fractions and used for RT-PCR. (c) PCR for P27 and HPRT on the input (I), supernatant (S) and immunoprecipitation (RIP) fractions. ES cells treated with (+) and without (-) dox were used. Each fraction of the ES cells with and without dox treatment was used to generate cDNA. The RT: + represents presence of Reverse Transcriptase and the RT: - represents no Reverse Transcriptase (control) during cDNA synthesis. (d) P27 levels in input fraction of untreated (dox-) and treated (dox+) cells when higher PCR cycle numbers (35 cycles) are used. (e) (top) Immunoblotting for HA-DND1 in the mRNP lysates, input and supernatant fraction of ES cells treated with (+) or without (-) dox. Control (C) was 293T cells transfected with HA-DND1 encoding expression vector. (bottom) Blots were reprobed with GAPDH. Relative HA-DND1 levels (HA-DND1/GAPDH ratios) in the fractions are indicated at the bottom in blue.

Figure 3

(a) Outline of RIP-RT and controls. hES/HA-DND1 cells were treated with dox (+DOX) or not treated with dox (-DOX). The cells were used to make mRNP lysates and the input fraction was incubated with anti-HA antibody or as control, anti-FLAG antibody for RIP. Excess tRNA was added to the input. RNA extracted from the RIP fraction was used for RT-PCR. (b) Defining the pluripotency factors which are targets of DND1. Anti-HA antibody (a-H) or anti-FLAG (a-F) was used for RIP. After RIP, RT-PCR was performed on equal amounts of cDNA from I, S and RIP fractions. RT+ indicates presence of Reverse Transcriptase during cDNA preparation. RT- are control lanes with no Reverse Transcriptase. Specific pull down of OCT4, SOX2 and NANOG by anti-HA antibody is observed in lane 13. The PCR cycle number used to detect each transcript is described in Methods. (c) Transcripts encoding pluripotency factors and cell cycle regulators targets are targets of DND1. RT-PCR of the S fraction was not performed on these samples. Specific pull down of LIN28, TP53 and LATS2 by anti-HA antibody is observed in lane 7. (d) Pro-apototic factor BAX and anti-apoptotic factor BCLX are specifically pulled down with HA-DND1 during RIP using anti-HA antibodies (lane 7).