We show that, in Xenopus laevis oocytes and early embryos, double-stranded exogenous siRNAs cannot function as microRNA (miRNA) mimics in either deadenylation or guided mRNA cleavage (RNAi). Instead, siRNAs saturate and inactivate maternal Argonaute (Ago) proteins, which are present in low amounts but are needed for Dicer processing of pre-miRNAs at the midblastula transition (MBT). Consequently, siRNAs impair accumulation of newly made miRNAs, such as the abundant embryonic pre-miR-427, but inhibition dissipates upon synthesis of zygotic Ago proteins after MBT. These effects of siRNAs, which are independent of sequence, result in morphological defects at later stages of development. The expression of any of several exogenous human Ago proteins, including catalytically inactive Ago2 (Ago2mut), can overcome the siRNA-mediated inhibition of miR-427 biogenesis and function. However, expression of wild-type, catalytically active hAgo2 is required to elicit RNAi in both early embryos and oocytes using either siRNA or endogenous miRNAs as guides. The lack of endogenous Ago2 endonuclease activity explains why these cells normally are unable to support RNAi. Expression of catalytically active exogenous Ago2, which appears not to perturb normal Xenopus embryonic development, can now be exploited for RNAi in this vertebrate model organism.

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro. Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

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Although Xenopus laevis is a powerful model organism that has provided critical mechanistic insights into vertebrate development, cell biology and neural biology its utility has been limited by a lack of genetic analysis and gene manipulation. Other organisms that are difficult to manipulate genetically have often been studied by the use of RNAi, the inactivation of specific genes through the use of small interfering RNAs (siRNAs) that target the mRNAs of these genes for degradation. However, RNAi does not work in Xenopus oocytes or early embryos, making it impossible to use siRNAs in the analysis of early stages of Xenopus development. Thus new methods or tools that would support the study of these events are greatly needed.Recently, we discovered that Xenopus oocytes and early embryos lack functional Ago2, the nuclease containing protein that is guided to targeted mRNAs by siRNAs, which would explain why these cells are unable to carry out RNAi. Fortunately, we have also found that exogenous Ago2 generated from injected in vitro synthesized mRNA overcomes this deficiency. Thus we can now perform RNAi in Xenopus oocytes and early embryos, potentially making them amenable to genetic analysis through inactivation of specific gene products.