Regulation of pou5f3 family pluripotency gene transcripts stability by Ybx1 ribonucleoprotein complexes in Xenopus laevis early development

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Abstract

Here, we studied the regulation of pou5f3 family transcripts stability by association with Ybx1, a protein of ribonucleoprotein complexes. It is known that the clawed frog Xenopus laevis has three genes belonging to the POU5 family: pou5f3.1/oct91, pou5f3.2/oct25, and pou5f3.3/oct60. The Pou5f3 family factors are orthologues of the mammalian embryonic stem cell OCT4 pluripotency factor. However, the expression patterns of these genes differ over time. Pou5f3.3/oct60 transcripts are stored in oocytes, are present in large quantities in fertilized eggs, and then degrade only after fertilization. Pou5f3.2/oct25 transcripts are also present in the zygote, but their numbers increase even more during the development process. Finally, pou5f3.1/oct91 transcription begins only after the activation of the embryo genome at the middle blastula stage. In the present work, we revealed a much higher specificity of the Ybx1 factor to form a complex with the maternal mRNA of the pou5f3.3/oct60 gene compared to zygotic mRNAs of the pou5f3.1/oct91 and pou5f3.2/oct25 genes. Since Ybx1 is a protein that, on the one hand, is involved in interaction with cytoskeletal proteins, and, on the other hand, binds and stabilizes pluripotency genes mRNA, it can play a linking role in between the degradation of these maternal transcripts and cytoskeletal rearrangements during the onset of morphogenetic cell movements in the process of formation of germ layers.

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About the authors

Е. А. Parshina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. G. Zaraisky

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997ul. Miklukho-Maklaya 16/10, Moscow, 117997

N. Y. Martynova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Author for correspondence.
Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Effect of Ybx1 on the amount of mRNA of the pou5f3 family genes in clawed frog embryos. (a) Scheme of the experiment to study the role of Ybx1 overexpression. (b) Expression level of injected myc-Ybx1 and myc-C-Ybx1 proteins shown by Western blotting with anti-myc antibodies. Tubulin detected by anti-tubulin monoclonal antibodies was used as a reference control. (c) Change in the expression level of the pou5f3 family genes in response to myc-Ybx1 and myc-C-Ybx1 overexpression revealed by qRT-PCR. Data are presented as a fold change in gene expression in the experimental embryos compared to the expression in the control embryos. (g) Schematic diagram of the experiment to study the role of Ybx1 knockdown by injection of morpholino oligonucleotides. (e) Western blotting with anti-Ybx1 antibodies from lysates of injected embryos; anti-tubulin monoclonal antibodies were used as a control. (e) Changes in the expression level of the pou5f3 family genes in response to Ybx1 knockdown and restoration of its normal expression by co-injection of myc-Ybx1, detected by qRT-PCR. Data are presented as fold changes in gene expression in experimental embryos compared to expression in control embryos. In all cases, the odc1 and eef1a1 genes were used for data normalization; standard deviations obtained from three independent experiments are shown.

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3. Fig. 2. (a) – Change in the expression level of the pou5f3 family genes in response to Ybx1 knockdown under actinomycin D transcription blocking conditions, detected by qRT-PCR. Data are presented as a fold change in gene expression in the experimental embryos compared to the expression in the control embryos. qRT-PCR of the housekeeping gene odc1 mRNA was used for data normalization. (b) – The level of mRNA of the pou5f3 family genes precipitated by myc-Ybx1 and myc-C-Ybx1 proteins in RNA immunoprecipitation experiments. Data are presented as a percentage of bound mRNA to the total amount of this mRNA in the lysate. In all cases, standard deviations obtained from three independent experiments are shown.

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