Celf1 and Tia1 are novel components of vegetal localization complexes

3.1.1. Celf1 and Tia1 form one complex with known vegetal localization factors

Celf1 and Tia1 were identified to interact with a vegetally localizing RNA inXenopusoocytes.

In order to verify the association of Celf1 and Tia1 with vegetal localization RNPs, Flag-tagged Celf1 and Tia1 proteins were expressed in transport competent stage III/IV oocytes and immuno-precipitated from corresponding oocyte extracts. Like the previously characterized vegetal localization factor Ptbp1 (Cote et al., 1999), Celf1 and Tia1 co-precipitate the known localization factors Stau1, Igf2bp3, Elavl1/2 and Hnrnpab (Figure 3.1).

Thus, Celf1 and Tia1 are indeed components of vegetal localization RNPs in Xenopus oocytes. RNAse treatment of corresponding oocyte extracts abolished co-precipitation of known localization factors (Figure 3.1), indicating that Celf1 and Tia1 interact with these known localization factors via an RNA scaffold.

Figure 3.1. Celf1 and Tia1 co-precipitate known localization RNP complex components inXenopusoocytes.Western blot analysis for co-precipitation of known localization complex components with Flag-tagged Tia1, Celf1 and Ptbp1 in the absence or presence of RNase.

Extracts of uninjected oocytes served as negative controls. The asterisk indicates an unspecific protein band caused by cross reactions of the anti-Flag antibody

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3.1.2. Celf1 and Tia1 are predominantly located in the cytoplasm and co-localize with dnd1-LE at the vegetal cortex

In order to analyze endogenous protein levels of Celf1 and Tia1 during oogenesis, extracts of staged oocytes were analyzed by Western blot using Celf1- or Tia1-antibodies. Endogenous Celf1 is detected throughout oogenesis and embryogenesis (Figure 3.2A). The reduced electrophoretic mobility of Celf1 after oocyte maturation has been reported to be caused by Celf1 phosphorylation (Detivaud et al., 2003). However, Celf1 protein bands appear as doublets throughout oogenesis and embryogenesis (Figure 3.2A). Phosphatase treatment of stage III and VI oocyte extracts abolished the slower migrating band (Figure S5), indicating that a fraction of Celf1 might be partially phosphorylated throughout oogenesis and embryogenesis. Two isoforms of Tia1 are generated by alternative splicing, Tia1a and Tia1b of 43 and 40 kDa, respectively (Beck et al., 1996; Figure 1.10). Both Tia1a and Tia1b are detected throughout oogenesis and embryonic development with highest protein levels during later stages of oogenesis and embryogenesis (Figure 3.2A).

Celf1 and Tia1 orthologs are detected in nuclei and cytoplasm of different cell types (Kedersha et al., 1999; Ladd and Cooper, 2004; Zhang et al., 2005). To examine the subcellular distributions of Celf1 and Tia1 in Xenopus oocytes, endogenous protein levels were analyzed in nuclear and cytoplasmic fractions of staged oocytes by Western blot. Celf1 is detected in the cytoplasm, but not in the nucleus, throughout oogenesis (Figure 3.2B). Tia1 is predominantly detected in cytoplasmic extracts with a minor fraction in the nuclear extracts of late stage oocytes (Figure 3.2B). Igf2bp3 has been shown to be predominantly localized in the cytoplasm (Loeber et al., 2010) and Hnrnpab to be enriched in the nucleus (Czaplinski et al., 2005). Thus, immuno-blots for Igf2bp3 and Hnrnpab served as loading controls for cytoplasmic and nuclear fractions, respectively (Figure 3.2B).

In order to analyze the intracellular distributions of Tia1 and Celf1 in oocytes in more detail, immunostaining of endogenous Celf1 and Tia1 in stage III albino oocytes was performed. To detect a potential co-localization of these proteins with vegetally localizing RNAs, oocytes were injected with Cy3-labeleddnd1-LE RNA prior to immunostaining and protein and RNA signals were analyzed by confocal microscopy. Cy3-dnd1-LE RNA is detected in granular structures in the vegetal cytoplasm and at the vegetal cortex (Figure 3.2C, D). Both Celf1 and Tia1 co-localize with Cy3-dnd1-LE-RNA at the vegetal cortex and in the more vegetally located granular structures (Figure 3.2C). Tia1 protein signals are additionally detected as small particles, which appear to be attached to the RNA granules (Figure 3.2C). Co-staining of Celf1 together with the localization factor Igf2bp3 revealed co-localization with

Cy3-dnd1-LE in the vegetal granules and at the vegetal cortex (Figure 3.2D). In summary, these results show that Celf1 and Tia1 are predominantly located in the cytoplasm and they co-localize with vegetally localizing RNA in Xenopus oocytes.

68 Results

Figure 3.2. Celf1 and Tia1 are predominantly cytoplasmic and co-localize withdnd1-LE at the vegetal cortex ofXenopus oocytes. A) Temporal analysis of endogenous Celf1 and Tia1 protein expression during Xenopusoogenesis and embryogenesis. Celf1 and Tia1 were detected by Western blot using equivalent amounts of oocyte, egg (E) and embryonic extracts of indicated stages. B) Analysis of nuclear/cytoplasmic distributions of Celf1 and Tia1 in Xenopusoocytes. Endogenous Tia1 and Celf1 were detected by Western blot using equivalent amounts of nuclear (N) and cytoplasmic (C) extracts of staged oocytes. Igf2bp3 and Hnrnpab served as controls for a predominantly cytoplasmic or nuclear protein, respectively. C) Analysis of intracellular protein distribution and co-localization of Celf1 and Tia1 with vegetal RNA.

Immunostainings of Celf1 and Tia1 were performed with stage III oocytes that were pre-injected with Cy3-dnd1-LE RNA. D) Analysis of Celf1 co-localization with the known localization factor Igf2bp3. Co-immunostaining of Celf1 and Igf2bp3 was performed with stage III oocytes that were pre-injected with Cy3-dnd1-LE RNA. Scale bars indicate 100 µm for whole oocytes and 20 µm for the magnified vegetal cortex.

3.2. Celf1 participates in vegetal localization of dnd1-LE