Co-IF in A6 cells

In contrast to the embryonic myocytes Seb4 protein is restricted to the cytoplasm in A6 cells. Nevertheless, it appeared very interesting to analyze the nuclear markers in this cellular background (Figure29).

Lamin serves as a marker for the nuclear envelope because its boundary is more precise than the one from the DAPI counterstain (Figure 29C). The other question if Seb4 is perhaps enriched inside or at the periphery around the nuclear envelope could be answered by analyzing the co-localization of Seb4 with lamin. The lamin dense region, seems devoid of Seb4 or at least of lower concentration.

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Figure 29: Co-localization of Seb4 protein and selected nuclear and cytoplasmic marker proteins in A6 cells. Co-IF of Seb4 protein with nuclear (C-E) and cytoplasmic (F) marker proteins. (A-B) Background controls, show secondary antibodies RRX donkey a-rat in red and Cy2 donkey a-mouse, and Cy2 donkey a-guinea pig. (A-F) DAPI stained DNA; (’) red channel shows Seb4 signal; (’’) green channel shows co-IF signal; (’’’) merge; (’’’’) detail of (’’’).

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Seb4 does not co-localize with lamin at the inside of the nuclear envelope, suggesting that Seb4 is localized outside the nucleus.

The antibodies against SF3b and DRSP mark the position of the splicing speckles in the nucleus (29D and E). As shown by the merges, the Seb4 protein signal does not overlap with the speckles markers, because it is located in the cytoplasmic compartment (29D andE).

In Figure 29F the nuclearplasm fluoresces via AND-1 detection. Additionally to the presence of the nucleus, the signal of AND-1 confirms, that the A6 cells do not undergo mitosis (see 3.5.4.2). Though the AND-1 signal is very weak, it is clear that AND-1 and DAPI overlap, whereas Seb4 is located to the cytoplasm.

Treacle protein is detected in Figure 29G. It is concentrated in the nucleolus as well as in these dotted nuclear entities. There is no Seb4 co-localization with Treacle. NO38 shows, besides its nucleolar localization a cytoplasmic distribution, where Seb4 is also localized (29H). Upon overlay of both signals, the cytoplasmic NO38 protein is located not directly adjacent to the nucleus, resulting in a gap of an exclusively Seb4-positive ring around the nucleus. Yet, the structure of the cytoplasmic NO38 does not correlate to the diffuse signal of Seb4 in the cytoplasm.

As mentioned above, Seb4 is localized in A6 cells primarily to the cytoplasm. So, it was very interesting to see if it co-localized with α-Tubulin (Figure

29I). In most cells the densities of both Seb4 and α-Tubulin signals are correlated. In

those cytoplasmic areas where Seb4 is enriched, a more concentrated α-Tubulin

structure (e.g. both cells at the top right) was detected. This result is confirmed by the yellow signal colour in the overlay. Nevertheless, Seb4 does not show the precise filamentous protein pattern, like α-Tubulin does.

Concluding from the co-localization experiments, nuclear Seb4 is not located to the nucleoli, splicing speckles, or sites of transcription. In regard to its cytoplasmic distribution, Seb4 does not appear to be associated with the cytoskeleton.

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4 Discussion

In the last decades, a large amount of developmental studies have been devoted to the identification and characterization of growth factors and transcription factors expressed in the early embryo. In the complex series of events involved in cellular differentiation and organogenesis, also post-transcriptional mechanisms constitute an additional layer of regulatory control over gene expression and are employed to modulate differentiation programmes regulated by growth and transcription factors. Indeed, a lot of evidence indicates that numerous developmental processes are regulated at the level of RNA processing, stability, localization, and translation by non-coding RNAs and/or RNA-binding proteins. MicroRNAs, a class of non-coding RNA molecules, function in potent inhibition of individual key targets or coordinated regulation of target clusters, fine-tuning of target activity, and the reversibility of some aspects of microRNA-mediated repression (Bushati & Cohen, 2007).

RBPs coordinate functionally related sets of mRNAs through binding with their RNA-binding domains to sequence elements in the mRNA. Considering the hundreds of RBPs encoded in eukaryotic genomes, post-transcriptional control may be even comparable in its richness and complexity to transcriptional regulatory systems. Although they are not generally as well characterized as signalling and transcriptional networks yet, the number of RBPs known today to be involved in vertebrate development is growing and provides an additional level of coordination.

The highly conserved, RRM containing protein Seb4 was described first in Xenopus laevis by Heinrich Jasper from our laboratory in 1998, as a direct target of MyoD (Jasper, 1998), and later by the Bouwmeester labratory (Fetka et al, 2000) as a tissue-specific putative RNA-binding protein identified in an RNA in situ screen for genes showing time-specific expression patterns. However, the role of Seb4 and its biochemical properties have not been published, yet.

In this study, I have characterized Seb4 in the early development of Xenopus laevis by various biological and biochemical approaches. Firstly, I analyzed the Seb4 mRNA and protein expression pattern and its subcellular localization. Next, I addressed the biological function of Seb4 revealing the skeletal and cardiac muscle differentiation pathways, in which Seb4 is involved, and the skeletal muscle and lens differentiation pathway, for which Seb4 is required. Thirdly, I attempted to identify RNA and protein interaction partners of Seb4 via RIP, IP and gelfiltration.

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