Abstract
BackgroundThe 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.ResultsES 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.ConclusionsOur 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.
Highlights
The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates
(dead end homolog 1) (DND1) is not detected in mouse embryo fibroblast (MEF) cells, the feeder cells on which the human embryonic stem cells (hES) cells grow
The hES/(hemagluttinin-tagged DND1) (HA-DND1) cell line uses the tetracycline on (Tet-On) system in which doxycycline turns on the expression of HA-DND1 [35,36] (Figure 1c)
Summary
The RNA binding protein, DEAD END (DND1), is essential for maintaining viable germ cells in vertebrates. It is a testicular germ cell tumor susceptibility factor in mice. Inactivation of the Dnd gene results in sterility in vertebrates as well as causes development of testicular germ cell tumors in mice [1,2]. Previous reports have shown that DND1 interacts with the 3’-untranslated region (UTR) of mRNAs such as that of the cell cycle inhibitor, P27 (p27Kip, CDKN1B) and cell cycle regulator and tumor suppressor, LATS2 (large tumor suppressor, homolog 2 of Drosophila - a serine/threonine-protein kinase) [11,13]. DND1 was shown to interact with the U-rich sequences on zebrafish Nanos and TDRD7 mRNAs
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