A novel role for Celf1 in vegetal RNA localization during Xenopus oogenesis.

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes. To identify ribosome-associated biogenesis factors for the OXPHOS system, we purified ribosomes and associated proteins from mitochondria. We identified TMEM223 as a ribosome-associated protein involved in complex IV biogenesis. TMEM223 stimulates the translation of COX1 mRNA and is a constituent of early COX1 assembly intermediates. Moreover, we show that SMIM4 together with C12ORF73 interacts with newly synthesized cytochrome b to support initial steps of complex III biogenesis in complex with UQCC1 and UQCC2. Our analyses define the interactome of the human mitochondrial ribosome and reveal novel assembly factors for complex III and IV biogenesis that link early assembly stages to the translation machinery.

Whole-Mount In Situ Hybridization of Xenopus Oocytes.

Whole-mount in situ hybridization (WMISH) is a common approach that is used to visualize spatial and temporal gene expression in embryos. In this process, digoxygenin-labeled antisense RNA is hybridized to the complementary transcript of interest and RNA hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. During Xenopus laevis oogenesis, certain RNAs localize to the animal or vegetal pole laying the foundation for germ cell development and germ layer formation of the future embryo. Here we present a WMISH protocol for Xenopus laevis oocytes allowing for the efficient detection of localized RNAs in a large number of oocytes during different stages of oogenesis. The application of this approach might be combined with microinjection of tagged reporter RNAs and/or a gain- or loss-of-function background, allowing for the functional analysis of single protein factors involved in RNA localization.

T-cell internal antigen 1 counteracts somatic RNA degradation during early Xenopus embryogenesis.

In Xenopus laevis, maternal transcripts that localize to the vegetal cortex of the oocyte are specifically inherited by prospective germ cells during cleavage stages. While a large fraction of maternal transcripts is degraded during the maternal to zygotic transition (MZT), transcripts associated with the germ-line are stable. A sequence in the dead end 1 3'UTR mediates vegetal localization in the oocyte as well as miR mediated clearance in somatic cells and germ cell specific stabilization during the MZT in embryos. We could identify Tia1 to co-precipitate with known components of vegetal localization RNPs in X. laevis oocytes. Tia1 interacts and co-localizes with various localization elements from vegetally localizing RNAs. In X. laevis embryos, ectopic expression of Tia1 counteracts somatic degradation of dnd1 localization element reporter RNAs and it can synergize with Dnd1 protein in reporter RNA stabilization. Ectopic Tia1 also protects several endogenous localizing and germ cell specific mRNAs from somatic degradation. Thus, proteins that protect germ-line transcripts from miR mediated decay during the MZT in embryos might bind these RNAs already in the oocyte.