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A reverse transcriptase-dependent mechanism is essential for murine preimplantation development.

Sciamanna I, Vitullo P, Curatolo A, Spadafora C - Genes (Basel) (2011)

Bottom Line: These data indicate an early requirement for LINE-1-encoded RT to support early developmental progression.Consistent with this, recent findings indicate that a reverse transcription wave is triggered in the zygote a few hours after fertilization and is propagated at least through the first two rounds of cell division.On the whole these findings suggest that reverse transcription is strictly required in early embryos as a key component of a novel RT-dependent mechanism that regulated the proper unfolding of the developmental program.

View Article: PubMed Central - PubMed

Affiliation: Italian National Institute of Health (ISS), Viale Regina Elena 299, 00161 Rome, Italy. ilaria.sciamanna@iss.it.

ABSTRACT
LINE-1 (Long Interspersed Nuclear elements) and HERVs (Human Endogenous Retroviruses) are two families of retrotransposons which together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly that encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is up-regulated in embryonic tissues and transformed cells. Here we review evidence indicating that the LINE-1-encoded RT plays regulatory roles in early embryonic development. Indeed, antisense-mediated inhibition of expression of a highly expressed LINE-1 family in mouse zygotes caused developmental arrest at the two- or four-cell embryo stages. Development is also arrested when the embryo endogenous RT activity is pharmacologically inhibited by nevirapine, an RT inhibitor currently employed in AIDS treatment. The arrest of embryonic development is irreversible even after RT inhibition is removed and it is associated with subverted gene expression profiles. These data indicate an early requirement for LINE-1-encoded RT to support early developmental progression. Consistent with this, recent findings indicate that a reverse transcription wave is triggered in the zygote a few hours after fertilization and is propagated at least through the first two rounds of cell division. On the whole these findings suggest that reverse transcription is strictly required in early embryos as a key component of a novel RT-dependent mechanism that regulated the proper unfolding of the developmental program.

No MeSH data available.


Related in: MedlinePlus

Microinjection of LINE-1-specific antisense oligonucleotides arrests embryo development at preimplantation stages. (a) Map of a murine full-length LINE-1/L1 element. The position of LINE-1 antisense oligonucleotide used by Beraldi et al. (2006) is symbolized by the arrow near the 5′ UTR; (b) Developmental progression in groups of mouse embryos treated as follows: non-injected (non inj, brown line), or injected with PBS (purple line), nonsense oligonucleotide (NS, green line) and L1 antisense oligonucleotide (anti-L1, orange line). Data represent mean values, and bars the standard deviation, from two to five experiments; (c) Functional assay of the endogenous RT activity (MS2 phage RNA was used as template and embryo lysates as the RT source). Lysates were either from nonsense oligonucleotide-injected embryos (ns), or from arrested embryos injected with L1 antisense (anti-L1). Negative control reactions included no lysate, no RNA or no primers, while positive controls utilized commercial RT. Retrotranscribed MS2 cDNA products were quantified by densitometry. Mean and standard deviations were calculated from three experiments and are expressed relative to values obtained in positive controls (taken as 100%) (Modified from Beraldi et al., 2006).
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f2-genes-02-00360: Microinjection of LINE-1-specific antisense oligonucleotides arrests embryo development at preimplantation stages. (a) Map of a murine full-length LINE-1/L1 element. The position of LINE-1 antisense oligonucleotide used by Beraldi et al. (2006) is symbolized by the arrow near the 5′ UTR; (b) Developmental progression in groups of mouse embryos treated as follows: non-injected (non inj, brown line), or injected with PBS (purple line), nonsense oligonucleotide (NS, green line) and L1 antisense oligonucleotide (anti-L1, orange line). Data represent mean values, and bars the standard deviation, from two to five experiments; (c) Functional assay of the endogenous RT activity (MS2 phage RNA was used as template and embryo lysates as the RT source). Lysates were either from nonsense oligonucleotide-injected embryos (ns), or from arrested embryos injected with L1 antisense (anti-L1). Negative control reactions included no lysate, no RNA or no primers, while positive controls utilized commercial RT. Retrotranscribed MS2 cDNA products were quantified by densitometry. Mean and standard deviations were calculated from three experiments and are expressed relative to values obtained in positive controls (taken as 100%) (Modified from Beraldi et al., 2006).

Mentions: To substantiate these results and rule out possible artifacts caused by non-specific off-target effects of nevirapine, we designed a second set of experiments to specifically down-regulate a highly expressed LINE-1 family in murine cells [52]. That LINE-1 family is regarded as the major, if not unique, source of RT activity responsible for most retrotransposition events in murine cells. We used an antisense oligonucleotide targeting the 5′-end of ORF1 in the murine LINE-1/L1 element (Figure 2A). LINE-1 antisense oligonucleotide microinjection in zygotes caused a total developmental arrest (Figure 2B), reproducing that induced by nevirapine [53]. Concomitant with this, the endogenous RT activity was again significantly reduced in arrested compared to control embryos at the same stage (Figure 2C). In contrast, microinjection of non-specific oligonucleotide of same length but scrambled sequence had no developmental consequence. These results provide a proof-of-principle that the endogenous RT activity is required for progression of early cleavage embryos and identify an active LINE-1 family as a major source of RT activity. In synthesis, therefore, both the sperm-derived and the embryo newly synthesized RT pools are strictly required for preimplantation development.


A reverse transcriptase-dependent mechanism is essential for murine preimplantation development.

Sciamanna I, Vitullo P, Curatolo A, Spadafora C - Genes (Basel) (2011)

Microinjection of LINE-1-specific antisense oligonucleotides arrests embryo development at preimplantation stages. (a) Map of a murine full-length LINE-1/L1 element. The position of LINE-1 antisense oligonucleotide used by Beraldi et al. (2006) is symbolized by the arrow near the 5′ UTR; (b) Developmental progression in groups of mouse embryos treated as follows: non-injected (non inj, brown line), or injected with PBS (purple line), nonsense oligonucleotide (NS, green line) and L1 antisense oligonucleotide (anti-L1, orange line). Data represent mean values, and bars the standard deviation, from two to five experiments; (c) Functional assay of the endogenous RT activity (MS2 phage RNA was used as template and embryo lysates as the RT source). Lysates were either from nonsense oligonucleotide-injected embryos (ns), or from arrested embryos injected with L1 antisense (anti-L1). Negative control reactions included no lysate, no RNA or no primers, while positive controls utilized commercial RT. Retrotranscribed MS2 cDNA products were quantified by densitometry. Mean and standard deviations were calculated from three experiments and are expressed relative to values obtained in positive controls (taken as 100%) (Modified from Beraldi et al., 2006).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3924816&req=5

f2-genes-02-00360: Microinjection of LINE-1-specific antisense oligonucleotides arrests embryo development at preimplantation stages. (a) Map of a murine full-length LINE-1/L1 element. The position of LINE-1 antisense oligonucleotide used by Beraldi et al. (2006) is symbolized by the arrow near the 5′ UTR; (b) Developmental progression in groups of mouse embryos treated as follows: non-injected (non inj, brown line), or injected with PBS (purple line), nonsense oligonucleotide (NS, green line) and L1 antisense oligonucleotide (anti-L1, orange line). Data represent mean values, and bars the standard deviation, from two to five experiments; (c) Functional assay of the endogenous RT activity (MS2 phage RNA was used as template and embryo lysates as the RT source). Lysates were either from nonsense oligonucleotide-injected embryos (ns), or from arrested embryos injected with L1 antisense (anti-L1). Negative control reactions included no lysate, no RNA or no primers, while positive controls utilized commercial RT. Retrotranscribed MS2 cDNA products were quantified by densitometry. Mean and standard deviations were calculated from three experiments and are expressed relative to values obtained in positive controls (taken as 100%) (Modified from Beraldi et al., 2006).
Mentions: To substantiate these results and rule out possible artifacts caused by non-specific off-target effects of nevirapine, we designed a second set of experiments to specifically down-regulate a highly expressed LINE-1 family in murine cells [52]. That LINE-1 family is regarded as the major, if not unique, source of RT activity responsible for most retrotransposition events in murine cells. We used an antisense oligonucleotide targeting the 5′-end of ORF1 in the murine LINE-1/L1 element (Figure 2A). LINE-1 antisense oligonucleotide microinjection in zygotes caused a total developmental arrest (Figure 2B), reproducing that induced by nevirapine [53]. Concomitant with this, the endogenous RT activity was again significantly reduced in arrested compared to control embryos at the same stage (Figure 2C). In contrast, microinjection of non-specific oligonucleotide of same length but scrambled sequence had no developmental consequence. These results provide a proof-of-principle that the endogenous RT activity is required for progression of early cleavage embryos and identify an active LINE-1 family as a major source of RT activity. In synthesis, therefore, both the sperm-derived and the embryo newly synthesized RT pools are strictly required for preimplantation development.

Bottom Line: These data indicate an early requirement for LINE-1-encoded RT to support early developmental progression.Consistent with this, recent findings indicate that a reverse transcription wave is triggered in the zygote a few hours after fertilization and is propagated at least through the first two rounds of cell division.On the whole these findings suggest that reverse transcription is strictly required in early embryos as a key component of a novel RT-dependent mechanism that regulated the proper unfolding of the developmental program.

View Article: PubMed Central - PubMed

Affiliation: Italian National Institute of Health (ISS), Viale Regina Elena 299, 00161 Rome, Italy. ilaria.sciamanna@iss.it.

ABSTRACT
LINE-1 (Long Interspersed Nuclear elements) and HERVs (Human Endogenous Retroviruses) are two families of retrotransposons which together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly that encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is up-regulated in embryonic tissues and transformed cells. Here we review evidence indicating that the LINE-1-encoded RT plays regulatory roles in early embryonic development. Indeed, antisense-mediated inhibition of expression of a highly expressed LINE-1 family in mouse zygotes caused developmental arrest at the two- or four-cell embryo stages. Development is also arrested when the embryo endogenous RT activity is pharmacologically inhibited by nevirapine, an RT inhibitor currently employed in AIDS treatment. The arrest of embryonic development is irreversible even after RT inhibition is removed and it is associated with subverted gene expression profiles. These data indicate an early requirement for LINE-1-encoded RT to support early developmental progression. Consistent with this, recent findings indicate that a reverse transcription wave is triggered in the zygote a few hours after fertilization and is propagated at least through the first two rounds of cell division. On the whole these findings suggest that reverse transcription is strictly required in early embryos as a key component of a novel RT-dependent mechanism that regulated the proper unfolding of the developmental program.

No MeSH data available.


Related in: MedlinePlus