The ribonucleoprotein complex factor Ybx1 stabilizes the maternal mRNA of the ssx2ip gene encoding the centrosome maturation protein in Xenopus laevis embryogenesis

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Abstract

Our study investigates the mechanisms that regulate early developmental gene expression in Xenopus laevis frog embryos. Our previous study demonstrated that maternal mRNAs of two developmentally significant genes, the nuclear retinoic acid receptor rxrg and the pluripotency factor pou5f3, form complexes with ribonucleoprotein complex Ybx1. Based on the results of the present study, we determined and demonstrated that the stability of the maternal mRNA ssx2ip, which encodes a conserved protein, also called Msd1 or ADIP, which is involved in centrosome maturation, is dependent on Ybx1. This research shows that Ybx1 forms a ribonucleoprotein complex with ssx2ip mRNA, which is mediated by its cold shock domain (CSD). This study confirms our hypothesis of Ybx1 selectively binding to maternal transcripts. It opens up new opportunities to study new mechanisms of gene expression regulation at the earliest stages of development by searching for possible cis-motifs for recognition by trans-regulators such as Ybx1.

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About the authors

E. A. Parshina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

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

A. G. Zaraisky

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

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

N. Y. Martynova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

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

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Supplementary files

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2. Fig. 1. (a) – Expression profile of the maternal ssx2ip gene (L-homolog – green, S-homolog – blue) by developmental stages of the frog X. laevis (according to Xenbase); (b) – scheme of the experiment on microinjections into X. laevis embryos; (c) – the effect of exogenous proteins 6Myc-Ybx1 and 6Myc-C-Ybx1 on the mRNA level of the ssx2ip gene in comparison with the previously discovered [17, 18] genes pou5f3.3 and rxrg, revealed by RT-PCR (* p < 0.05, ns – statistically insignificant); (d) – the effect of translation suppression by morpholine oligonucleotides (MO) of ybx1 mRNA on the amount of mRNA of the maternal genes pou5f3.3, rxrg, ssx2ip and restoration of the normal level of these transcripts by injection of synthetic RNA 6myc-ybx1, revealed by RT-PCR (* p < 0.01, ns – statistically insignificant); (e) – the level of ssx2ip mRNA in response to microinjections of synthetic mRNA 6myc-ybx1 under conditions of transcription blocking with actinomycin D, revealed by RT-PCR (* p < 0.05, ns – statistically insignificant). RT-PCR with mRNA of the housekeeping gene odc1 was used for data normalization. All the above data revealed by the RT-PCR method are presented as a fold change in gene expression in the experimental embryos compared to the expression in the control embryos. In all cases, the odc1 and eef1a1 genes were used for data normalization; standard deviations obtained as a result of three independent experiments are shown; (e) RNA immunoprecipitation results: precipitation of ssx2ip mRNA with factors 6Myc-Ybx1 and 6Myc-C-Ybx1 in comparison with the previously studied rxrg and pou5f3.3 mRNAs [17, 18] (* p < 0.01, ns – statistically insignificant). Resin with anti-Flag antibodies was used as a control. Data are presented as a percentage of bound mRNA to the total amount of a given mRNA in the lysate. In all cases, standard deviations obtained as a result of three independent experiments are shown; (g) – effect of suppression of mRNA translation of the cytoskeletal protein zyxin by morpholine oligonucleotides (* p < 0.05, ns – statistically insignificant). The amount of ssx2ip mRNA decreases, while the level of pou5f3.3 and rxrg mRNA increases in response to knockdown of the zyxin gene.

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