Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in <i>Xenopus laevis</i>

Lund, Elsebet; Sheets, Michael; Imboden, Susanne Blaser; Dahlberg, James E.

doi:10.1101/gad.2038811

Cited by 80 publications

(90 citation statements)

References 70 publications

(94 reference statements)

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“…Alternatively, if artificially synthesized piRNA precursors (Kawaoka et al 2011) or piRNA mimics of 23-30 nt in length are effectively introduced by transfection or microinjection into cells with an active piRNA pathway, they could also cause specific silencing. The piRNA-mediated gene knockdown might also be useful in early embryos or oocytes of some animals such as Xenopus since RNAi and miRNA biogenesis are restricted in these species due to the limited amount of the Argonaute proteins (Lund et al 2011). …”

Section: Discussionmentioning

confidence: 99%

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

et al. 2012

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In germ cells, early embryos, and stem cells of animals, PIWI-interacting RNAs (piRNAs) have an important role in silencing retrotransposons, which are vicious genomic parasites, through transcriptional and post-transcriptional mechanisms. To examine whether the piRNA pathway can be used to silence genes of interest in germ cells, we have generated knock-in mice in which a foreign DNA fragment was inserted into a region generating pachytene piRNAs. The knock-in sequence was transcribed, and the resulting RNA was processed to yield piRNAs in postnatal testes. When reporter genes possessing a sequence complementary to portions of the knock-in sequence were introduced, they were greatly repressed after the time of pachytene piRNA generation. This repression mainly occurred at the post-transcriptional level, as degradation of the reporter RNAs was accelerated. Our results show that the piRNA pathway can be used as a tool for sequence-specific gene silencing in germ cells and support the idea that the piRNA generating regions serve as traps for retrotransposons, enabling the host cell to generate piRNAs against active retrotransposons.

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Section: Discussionmentioning

confidence: 99%

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

et al. 2012

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“…The enzyme dicer that cleaves pre-miRNA into mature miRNA, have only one fourth of the activity in X. laevis oocytes compared to matured cells (Lund et al, 2011;Muggenhumer, 2010;Muggenhumer et al, 2014). Therefore, the Na channel activity is only suppressed by 58 % in oocytes expressing miRNA-SCN2A ( figure 1c in paper III).…”

Section: Resultsmentioning

confidence: 98%

“…However, in X. laevis oocytes, cDNA plasmids expressing premiRNA need to be injected into the nucleus for it to be transcribed ( Fig. 3) (Lund et al, 2011). In the nucleus, pre-miRNA is bound to exportin 5 (EXP5) and cofactor Ran that has GTP bound to it.…”

Section: Mirna Expressionmentioning

confidence: 99%

The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes

Englund¹

2014

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Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration

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“…A recent study suggests that this is due to a lack of the Argonaute-2 (Ago2) protein in embryos. Ago2 is necessary for dicer dependent nuclease activity and Lund et al have shown that expression of exogenous Ago2 is sufficient for RNAi activity in Xenopus (Lund et al, 2011). However, for many years RNAi seemed impossible in Xenopus; therefore, studies in Xenopus have made use of synthetic nonnatural oligonucleotides to target RNA activity, summarized in Table 4.…”

Section: Controlling Translation With Small Moleculesmentioning

confidence: 99%

Xenopus: An ideal system for chemical genetics

Wheeler

Liu

2012

Genesis

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Summary: Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches. genesis 50: 207-218, 2012. V V C 2012 Wiley Periodicals, Inc.

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Limiting Ago protein restricts RNAi and microRNA biogenesis during early development in Xenopus laevis

Cited by 80 publications

References 70 publications

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

The role of ion channels and intracellular metal ions in apoptosis of Xenopus oocytes

Xenopus: An ideal system for chemical genetics

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