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Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development.

Ohnishi Y, Totoki Y, Toyoda A, Watanabe T, Yamamoto Y, Tokunaga K, Sakaki Y, Sasaki H, Hohjoh H - Nucleic Acids Res. (2010)

Bottom Line: Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs).Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes.Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Neuroscience, NCNP, Tokyo, Japan.

ABSTRACT
Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8-16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.

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Active small RNAs derived from retrotransposons. (A) Small RNAs (19–24 nt) of the plus strand (red) and minus strand (blue) matched the MERVL-Mm retrotransposon in mouse MII oocytes and pre-implantation embryos. Hatched bar represents an insertion into the GFP 3′ UTR. (B and C) Degradation of GFP fusion mRNA carrying MERVL-Mm. The GFP, GFP-Actin, GFP-MERVL-Mm (sense), or GFP-MERVL-Mm (antisense) mRNAs was introduced together with the DsRed mRNA as a control into 1-cell and 8–16-cell stage embryos by electroporation. After 0.5 and 18 h of electroporation, total RNA was extracted from 5 to 10 embryos and the introduced mRNAs were quantified by Q-PCR. The levels of GFP and its fusion mRNAs were normalized to those of DsRed mRNAs, and then further normalized to those obtained at 0.5 h in each case [n = 3; error bars represent SEM; *P < 0.05 (t-test)].
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Figure 2: Active small RNAs derived from retrotransposons. (A) Small RNAs (19–24 nt) of the plus strand (red) and minus strand (blue) matched the MERVL-Mm retrotransposon in mouse MII oocytes and pre-implantation embryos. Hatched bar represents an insertion into the GFP 3′ UTR. (B and C) Degradation of GFP fusion mRNA carrying MERVL-Mm. The GFP, GFP-Actin, GFP-MERVL-Mm (sense), or GFP-MERVL-Mm (antisense) mRNAs was introduced together with the DsRed mRNA as a control into 1-cell and 8–16-cell stage embryos by electroporation. After 0.5 and 18 h of electroporation, total RNA was extracted from 5 to 10 embryos and the introduced mRNAs were quantified by Q-PCR. The levels of GFP and its fusion mRNAs were normalized to those of DsRed mRNAs, and then further normalized to those obtained at 0.5 h in each case [n = 3; error bars represent SEM; *P < 0.05 (t-test)].

Mentions: When GFP RNAs carrying the MERVL-Mm sequences (Figure 2A) were introduced into one-cell and 8–16-cell embryos, the level of the GFP RNAs carrying the MERVL-Mm sense-strand sequence was markedly decreased in the 8–16-cell embryos, and slightly reduced in the one-cell embryos (Figure 2B and C). The GFP RNAs with the MERVL-Mm antisense-strand sequence was unaffected and slightly affected in one-cell and 8–16-cell stage embryos, respectively. Thus, our data indicate a stage-specific silencing of MERVL-Mm in early developing mouse embryos, and the silencing is likely dependent upon the level of the siRNAs derived from the MERVL-Mm retrotransposon itself (Figure 2A and Supplementary Figure S2A and C). In addition, Dicer-knockdown embryos (Figure 3A) showed a marked increase in the level of the MERVL-Mm transcript, consistently suggesting the involvement of RNAi in the MERVL-Mm retrotransposon silencing [ref. (31), Figure 3B].Figure 2.


Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development.

Ohnishi Y, Totoki Y, Toyoda A, Watanabe T, Yamamoto Y, Tokunaga K, Sakaki Y, Sasaki H, Hohjoh H - Nucleic Acids Res. (2010)

Active small RNAs derived from retrotransposons. (A) Small RNAs (19–24 nt) of the plus strand (red) and minus strand (blue) matched the MERVL-Mm retrotransposon in mouse MII oocytes and pre-implantation embryos. Hatched bar represents an insertion into the GFP 3′ UTR. (B and C) Degradation of GFP fusion mRNA carrying MERVL-Mm. The GFP, GFP-Actin, GFP-MERVL-Mm (sense), or GFP-MERVL-Mm (antisense) mRNAs was introduced together with the DsRed mRNA as a control into 1-cell and 8–16-cell stage embryos by electroporation. After 0.5 and 18 h of electroporation, total RNA was extracted from 5 to 10 embryos and the introduced mRNAs were quantified by Q-PCR. The levels of GFP and its fusion mRNAs were normalized to those of DsRed mRNAs, and then further normalized to those obtained at 0.5 h in each case [n = 3; error bars represent SEM; *P < 0.05 (t-test)].
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Active small RNAs derived from retrotransposons. (A) Small RNAs (19–24 nt) of the plus strand (red) and minus strand (blue) matched the MERVL-Mm retrotransposon in mouse MII oocytes and pre-implantation embryos. Hatched bar represents an insertion into the GFP 3′ UTR. (B and C) Degradation of GFP fusion mRNA carrying MERVL-Mm. The GFP, GFP-Actin, GFP-MERVL-Mm (sense), or GFP-MERVL-Mm (antisense) mRNAs was introduced together with the DsRed mRNA as a control into 1-cell and 8–16-cell stage embryos by electroporation. After 0.5 and 18 h of electroporation, total RNA was extracted from 5 to 10 embryos and the introduced mRNAs were quantified by Q-PCR. The levels of GFP and its fusion mRNAs were normalized to those of DsRed mRNAs, and then further normalized to those obtained at 0.5 h in each case [n = 3; error bars represent SEM; *P < 0.05 (t-test)].
Mentions: When GFP RNAs carrying the MERVL-Mm sequences (Figure 2A) were introduced into one-cell and 8–16-cell embryos, the level of the GFP RNAs carrying the MERVL-Mm sense-strand sequence was markedly decreased in the 8–16-cell embryos, and slightly reduced in the one-cell embryos (Figure 2B and C). The GFP RNAs with the MERVL-Mm antisense-strand sequence was unaffected and slightly affected in one-cell and 8–16-cell stage embryos, respectively. Thus, our data indicate a stage-specific silencing of MERVL-Mm in early developing mouse embryos, and the silencing is likely dependent upon the level of the siRNAs derived from the MERVL-Mm retrotransposon itself (Figure 2A and Supplementary Figure S2A and C). In addition, Dicer-knockdown embryos (Figure 3A) showed a marked increase in the level of the MERVL-Mm transcript, consistently suggesting the involvement of RNAi in the MERVL-Mm retrotransposon silencing [ref. (31), Figure 3B].Figure 2.

Bottom Line: Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs).Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes.Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, National Institute of Neuroscience, NCNP, Tokyo, Japan.

ABSTRACT
Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8-16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.

Show MeSH