Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos

Svoboda, Petr

doi:10.1159/000078215

Cited by 33 publications

(23 citation statements)

References 97 publications

(157 reference statements)

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“…Therefore, variation in developmental capacity due to the microinjection procedure has been ruled out. Previous studies have shown that the mechanism of RNAi is limited at the post-transcriptional level by degrading the sequence-specific mRNA or blocking the activity of ribosomal RNA (rRNA) (Svoboda 2004), which leads to a loss of function in mice (Svoboda et al 2000, Wianny & Zernicka-Goetz 2000, Grabarek et al 2002, Siddall et al 2002. Our results also showed that the injection of dsRNA of oocyte-or zygote-specific transcripts induced sequence-specific mRNA degradation and prevented subsequent protein synthesis during development of preimplantation embryos.…”

Section: Discussionsupporting

confidence: 66%

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Nganvongpanit

Müller²,

Rings³

et al. 2006

Reproduction

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RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P!0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P!0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

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Section: Discussionsupporting

confidence: 66%

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Nganvongpanit

Müller²,

Rings³

et al. 2006

Reproduction

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“…However, this replacement was not equivalent to LGN reduction or depletion because LGN-N-GFP functions similarly to LGN at the spindle and endogenous LGN is still functional whether it is localized to the spindle or not, making the overexpression effect of LGN-N-GFP similar to that of LGN. Double-stranded RNA-mediated mRNA degradation is an effective approach to knock down a gene in mouse oocytes without inducing an apoptotic response [23,24,34]. Using this technique, we reduced the maternally stored LGN mRNA by more than 90% by 40 h. Although LGN protein has a low turnover rate and was only reduced by about 50%, dsRNA-mediated downregulation of LGN revealed its roles in meiotic spindle organization.…”

Section: Discussionmentioning

confidence: 99%

Pins homolog LGN regulates meiotic spindle organization in mouse oocytes

Guo

Gao

2009

Cell Res

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Mouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without conventional centrosomes, the meiotic spindle has less focused poles and is barrel-shaped. The migration of meiotic spindles to the cortex is accompanied by a local reorganization and polarization of the cortex. LGN is a conserved protein involved in cell polarity and regulation of spindle organization. In the present study, we characterized the localization dynamics of LGN during mouse oocyte maturation and analyzed the effects of LGN upregulation and downregulation on meiotic spindle organization. At the germinal vesicle stage, LGN is distributed both cytoplasmically and at the cortex. During maturation, LGN localizes to the meiotic spindle apparatus and cortical LGN becomes less concentrated at the actin cap region. Excessive LGN induces meiotic spindle organization defects by elongating the spindle and enhancing pole focusing, whereas depletion of LGN by RNA interference results in meiotic spindle deformation and chromosome misalignment. Furthermore, the N-terminus of LGN has the ability of full-length LGN to regulate spindle organization, whereas the C-terminus of LGN controls cortical localization and polarization. Our results reveal that LGN is cortically polarized in mouse oocytes and is critical for meiotic spindle organization.

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“…Today, long RNA hairpins are shadowed by short hairpin systems, and they are used only in special cases where a short hairpin RNA system cannot be used efficiently. Long hairpin RNA was successfully used to block gene function in several types of mammalian cells, but aside from mouse oocytes, it never acquired wider attention (reviewed in [139]). Long dsRNA expression from a large inverted repeat remains a common solution for transgenic RNAi approach in invertebrates and plants.…”

Section: Do Long Hairpin Rnas Naturally Occur In Mammals?mentioning

confidence: 99%

“…It can also be combined with tissue-specific pol II promoters. Working with inverted repeats may be complicated (reviewed in [139]), but despite all the possible pitfalls, transgenic RNAi in mouse oocytes has produced a functional knockdown in several instances [120,[140][141][142], and the list of successful knockdowns in Caenorhabditis and Drosophila is much longer.…”

Section: Do Long Hairpin Rnas Naturally Occur In Mammals?mentioning

confidence: 99%

Hairpin RNA: a secondary structure of primary importance

Svoboda

Cara

2006

Cell. Mol. Life Sci.

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191

157

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Abstract. An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of Cell. Mol. Life Sci. 63 (2006) 901-918 1420-682X/06/080901-18 DOI 10.1007/s00018-005-5558-5 © Birkhäuser Verlag, Basel, 2006 trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought.Keywords. dsRNA, miRNA, RNAi, stem-loop, hairpin, localization, translation. IntroductionAn RNA hairpin consists of a double-stranded RNA (dsRNA) stem, often containing mismatches and bulges (i.e. unpaired sequences within the stem), and a terminal loop. It is the most common secondary structure found in almost every RNA folding prediction. RNA hairpins originate by two mechanisms: (i) transcription by DNAdependent RNA polymerase of an inverted repeat DNA resulting in the RNA folding into a stem-loop structure, and (ii) an RNA molecule formed as a folded-back template for RNA-dependent RNA polymerase, which synthesizes the second strand of the stem. This review will focus exclusively on the first case -RNA hairpins folded within RNA transcripts. The second mechanism, which produces perfect long dsRNA hairpins, is not widespread in nature and is probably restricted to a 'copy-back' mechanism of replication in certain viruses [1]. It is difficult to classify RNA hairpins into distinct categories because these structures easily arise and differ in many aspects. Structurally, RNA hairpins can occur in different positions within different types of RNAs; they differ in the length of the stem, the size of the loop, the number and size of bulges, and in the actual nucleotide sequence (Fig. 1a). These parameters provide an extreme variability allowing specific interactions with proteins (discussed in detail in [2]). Functionally, RNA hairpins can regulate gene expression in cis or trans, i.e. an RNA hairpin within an RNA molecule can regulate just that molecule (cis) or it can induce effects on other RNAs or pathways (trans). Hairpins serve as binding sites for a variety of proteins, act as substrates for enzymatic reactions as well as display intrinsic enzymatic activities. Many of the pathways utilizing and/or responding to RNA hairpins have evolved independently and are not linked to others. It is not possible to pr...

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Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos

Cited by 33 publications

References 97 publications

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Pins homolog LGN regulates meiotic spindle organization in mouse oocytes

Hairpin RNA: a secondary structure of primary importance

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