Celf1 and Tia1 counteract degradation of localizing RNAs

3.3.1. Ectopic expression of Celf1 and Tia1 antagonizes somatic microRNA-mediated decay of dnd1-LE reporter RNA in Xenopus embryos

Previous studies showed that the dnd1-LE mediates, in addition to vegetal localization, the restriction of dnd1 transcripts to germ cells in Xenopus embryos (Koebernick et al., 2010).

This restriction is based on the miR-mediated somatic degradation and germ cell specific protection of the dnd1-LE. Strikingly, proteins that are components of vegetal localization complexes in oocytes are involved in the protection ofdnd1mRNA from somatic degradation in embryos (Arthur et al., 2009; Koebernick et al., 2010). In order to analyze if Celf1 and Tia1 are involved in the stabilization ofdnd1RNA in embryos, a wild-type dnd1-LE reporter RNA was either injected alone or together with RNA encoding Celf1 or Tia1 into 2-cell stage Xenopusembryos (Figure 3.10A,). Reporter RNA levels were analyzed after the maternal to zygotic transition (MZT) at stage 32 by whole mountin situ hybridization (Figure 3.10B, E).

While the dnd1-LE reporter RNA alone is degraded during MZT and only detectable in primordial germ cells (PGCs), ectopic expression of Celf1 or Tia1 leads to a somatic stabilization of the dnd1-LE reporter RNA (Figure 3.10B-E). This result indicates that Celf1 and Tia1 may function in the germ cell specificdnd1RNA protection in embryos. The mutCelf1

dnd1-LE reporter RNA contains mutations in the miR-430/18 binding site (Figure 3.4A) (Koebernick et al., 2010) that probably interfere with miR-mediated decay. Thus, overexpression of Celf1 in embryos that were injected with mut_Celf1 dnd1-LE reporter RNA, was not considered. The miR-430/18 binding site is not affected in mutTia1 dnd1-LE (Koebernick et al., 2010; Figure 3.4A). In order to control for the specificity of Tia1 mediated RNA stabilization, a reporter RNA that contains the mutTia1 dnd1-LE with reduced affinity for Tia1 was injected alone or together with RNA encoding Tia1 (Figure 3.10D). Although mutTia1

dnd1-LE reporter RNA is degraded in the soma (Figure 3.10E, F), it is stable in PGCs.

However, the signal of the mutTia1 dnd1-LE is slightly weaker as compared to the wild-type dnd1-LE reporter RNA (Figure 3.10E). This PGC specific stabilization might be caused by low affinity binding of Tia1 and Elavl2 (Figure 3.4C). Still, the mutTia1dnd1-LE-reporter RNA is not stabilized in the soma by Tia1 overexpression (Figure 3.10E, F), indicating that overexpression of Tia1 specifically protects target RNAs from somatic degradation. In summary, these experiments suggest that Celf1 and Tia1 counteract dnd1-LE-reporter RNA degradation in PGCs.

Figure 3.10. Ectopic expression of Celf1 and Tia1 stabilizes dnd1-LE reporter RNA in Xenopusembryos.A) A reporter RNA that comprises thegfpopen reading frame (ORF) and the wild-type (wt)dnd1-LE (dnd1-LE-R) was injected into both vegetal blastomeres of two-cell stage Xenopus embryos. At stage 32, embryos were analyzed by WMISH against gfp.

B) Embryos injected with only thednd1-LE reporter RNA (dnd1-LE-R) or co-injected with RNA encoding Celf1.dnd1-LE reporter RNA alone is degraded in the soma and stable in germ cells.

Co-expression of Celf1 leads to somatic stabilization of dnd1-LE reporter RNA.

C) Quantification ofdnd1-LE reporter RNA levels scored in the injected embryos. Mean values of three independent experiments are shown. Error bars indicate standard error of the mean.

D) Wild-type (wt) and mutant (mutTia1) dnd1-LE reporter RNAs were injected as described above. E) Embryos injected with wild-type (wt) or mutant (mutTia1) dnd1-LE reporter RNAs alone or co-injected with RNA encoding Tia1. Wild-type and mutant dnd1-LE reporter RNAs are degraded in the soma and stable in PGCs. Co-expression of Tia1 leads to somatic stabilization of wild-type dnd1-LE, but not of mutantdnd1-LE reporter RNA. F) Quantification of dnd1-LE reporter RNA levels (only in PGCs or stabilized in the endoderm) scored in the injected embryos. Mean values of two independent experiments are shown, error bars indicate standard error of the mean.

84 Results

3.3.2. Tia1 synergizes with Dnd1 in the stabilization of dnd1-LE reporter RNA in Xenopus embryos

tia1mRNA is mainly expressed outside of germ cells, e.g. in neuronal structures (Rothé et al., 2006). Thus, it is unclear how Tia1 is able to specifically protect dnd1-LE in PGCs.

However, tia1 transcripts were detected in Xenopus PGCs by RNA sequencing (Aliaksandr Dzementsei, unpublished), suggesting the presence of Tia1 proteins in PGCs. Thus, germ cell specific protection of RNAs by Tia1 might involve interactions with germ cell specific proteins. Elavl2 has been shown to synergize with the germ cell specific protein Dnd1 in germline specific RNA stabilization (Koebernick et al., 2010). To test wether Tia1 also synergizes with Dnd1 indnd1RNA stabilization, both proteins were co-expressed in embryos and dnd1-LE reporter RNA stability was analyzed by WMISH. Ectopic expression of low doses of either Tia1 or Dnd1 proteins do not or only weakly stabilize co-injected wild-type dnd1-LE reporter RNA in the soma (Figure 3.11A, B). But, if low doses of both proteins are co-expressed in embryos,dnd1-LE reporter RNA stability is highly increased (Figure 3.11A, B). Thus, Tia1 and Dnd1 proteins synergize in the protection of germline RNAs from miR-mediated decay.

Figure 3.11. Tia1 synergizes with Dnd1 in the stabilization of dnd1-LE reporter RNA in Xenopus embryos. A) Embryos injected with gfp-dnd1-LE reporter RNA (dnd1-LE-R wt) alone or co-injected with low doses of RNA encoding Tia1, Dnd1, or both. Reporter RNA levels were detected by WMISH at stage 32. Injected wild-type dnd1-LE reporter RNA alone is degraded in the soma and stable in PGCs. Co-expression of low doses of Tia1 or Dnd1 does not stabilize dnd1-LE reporter RNA in the soma. Co-expression of same doses of Tia1 and Dnd1 leads to somatic stabilization of dnd1-LE reporter RNA. B) Quantification of dnd1-LE reporter RNA levels scored in the injected embryos. Mean values of three independent experiments are shown, error bars indicate standard error of the mean.

3.3.3. Ectopic expression of Tia1 leads to the stabilization of several vegetally localizing and germ cell specific RNAs in Xenopus embryos

Tia1 interacts with thednd1-LE in vitro and mediates protection of adnd1-LE reporter RNA from miR-mediated decay in embryos (Figure 3.3, 3.10, 3.11). In order to analyze if Tia1 also interacts with other LE RNAs, an in vitro interaction assay was performed using in vitro translated Flag-tagged proteins and different Cy3-labeled LE RNAs (Figure 3.12A, B). Tia1 binds to the dnd1-LE, the grip2-LE and the gdf1-LE, while it does not bind to the velo1-LE and to the non-localizing control RNAß-globin-3'UTR (Figure 3.12A). Thisin vitro interaction assay suggests that Tia1 might also stabilize the corresponding mRNAs in embryos.

In order to analyze the effect of Tia1 overexpression on different other mRNAs, endogenous RNA levels were assayed after ectopic expression of Tia1 by nanostring multiplex analysis.

Whereas Tia1 overexpression did not affected RNA levels before MZT (stage 8) and only slightly during MZT (stage 11), several mRNA levels are highly increased after MZT (stage 14) (Figure 3.12C, Table S1-4), suggesting that Tia1 antagonizes miR-mediated decay during MZT. In concordance with RNA bindingin vitro (Figure 3.12A), endogenous dnd1,gdf1 and grip2 mRNAs show increased RNA levels in Tia1 overexpressing oocytes (Figure 3.12C).

Other localizing mRNAs includingpgat andvelo1 and the non-localizing, but miR regulated, RNAcyclin b2 (Lund et al., 2009) are not affected by Tia1 overexpression (Figure 3.12C).

The mRNAs that are stabilized upon Tia1 over-expression include the germ cell specific transcriptsgermes,dnd1,grip2andcpeb1a(Figure 3.12C), suggesting that Tia1 counteracts miR-mediated decay of germline RNAs. However,gdf1 mRNA levels are also increased up to three fold upon Tia1 overexpression, although this mRNA is not germ cell specific (Figure 3.12C). Thus, Tia1 might also stabilize RNAs which are not germ cell specific by counteracting other decay mechanisms that are not mediated by miRs.

86 Results

Figure 3.12. Tia1 binds to several LEsin vitroand ectopic expression of Tia1 leads to stabilization of vegetally localizing and germ cell specific RNAs inXenopusembryos.A) In vitro interaction analysis of Tia1 with different LE RNAs. Cy3-labeled LE RNAs orß-globin 3'UTR (control) were co-immunoprecipitated with in vitro translated Flag-tagged Tia1, Celf1 and Ptbp1 proteins. Unprogrammed reticulocyte lysate served as negative control (-). RNAs were separated by urea-PAGE and detected by fluorescence imaging. B) Expression control for in vitro translated Flag-tagged proteins used in A). Proteins were detected by anti-Flag Western blotting. C) Fold change RNA levels of different localizing and control mRNAs at indicated developmental stages after overexpression of Tia1 in embryos.Xenopus2-cell stage embryos were injected with 200 or 400 pgtia1RNA. Embryos were grown until stages 8, 11 or 14 and subjected to total RNA extraction. RNA samples were analyzed using the nCounter®

Gene Expression assay (NanoString Technologies). The averaged fold changes of selected genes over uninjected control embryos of two independent experiments are shown, error bars indicate standard error of the mean.