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. Author manuscript; available in PMC: 2014 Aug 1.
Published in final edited form as: Nat Genet. 2013 Dec 15;46(2):152–160. doi: 10.1038/ng.2853

Figure 3. The interaction between TDP-43 and Ataxin-2 is mediated through the polyA binding protein motif of Ataxin-2.

Figure 3

a) Schematic representation of the versions of Ataxin-2 generated and tested for interactions with TDP-43. Colored boxes indicate the putative RNA-binding Lsm domain, the Lsm-associated domain LsmAD, the PABP-interacting motif PAM2 and the polyQ stretch. Deletion region of ΔLSM is aa277-346, and ΔPAM2 is aa920-932 of ATXN2-32Q. Both domains are well conserved from yeast to mammal.

b) TDP-43 toxicity is not affected by co-expression of ATXN2 lacking the PAM2 motif. Whereas TDP-43 toxicity is enhanced with co-expression of ATXN2-32Q or ATXN2-32Q-ΔLSM (TDP-43+ATXN2-32Q or TDP-43+ΔLSM), there is no effect with co-expression of ATXN2-32Q-ΔPAM2 (TDP-43+ΔPAM2). Genotypes: YFP is gmr-GAL4(YH3)/UAS-YFP. TDP-43 is UAS-TDP-43(M)/+; gmr-GAL4(YH3)/+. TDP-43+ATXN2-32Q is UAS-TDP-43(M)/+; gmr-GAL4(YH3)/UAS-ATXN2-32Q. TDP-43+ΔPAM2 is UAS-TDP-43(M)/+; gmr-GAL4(YH3)/UAS-ATXN2-32Q.ΔPAM2. TDP-43+ΔLSM is UAS-TDP-43(M)/+; gmr-GAL4(YH3)/UAS- ATXN2-32Q.ΔLSM. Scale bar for eyes, 100 μm; for sections, 10 μm.

c) Expression of TDP-43 in the nervous system reduces lifespan (green, compared with normal in brown). Expression of ATXN2-32Q greatly enhanced TDP-43 toxicity (blue), but the ATXN2-32Q.ΔPAM2 deletion mutant form did not (red). Genotypes: Elav/+ is elav3A-GAL4/+ (n=177 flies). Elav, TDP-43/+ is elav3A-GAL4, UAS-TDP-43(S)/+ (n=156). Elav, TDP-43/ATXN2-32Q is elav3A-GAL4, UAS-TDP-43(S)/UAS-ATXN2-32Q (n=152). Elav, TDP-43/ΔPAM2 is elav3A-GAL4, UAS-TDP-43(S)/UAS-ATXN2-32Q. ΔPAM2 (n=138).

d) TDP-43 caused progressive loss of climbing ability when expressed in the adult nervous system (green) which is dramatically enhanced by co-expression of ATXN2-32Q (blue). However, the ΔPAM2 form of ATXN2-Q32 has little effect (red) compared to TDP-43 alone. Mean ± 95% CI of four experiments (n=125 flies per genotype). # p<0.0001, n.s., not significant (ANOVA for significance, followed by Tukey’s multiple comparison test).

e) Total, nuclear and cytosolic TDP-43 protein levels in 10d fly heads. Cytosolic TDP-43 protein level is greatly increased by ATXN2-32Q; ΔPAM2 expression does not affect TDP-43 localization or level. Mean ± s.e.m., n=3-4. # p<0.0001, n.s., not significant (ANOVA for significance, followed by Tukey’s multiple comparison test).

d–e Genotypes: elavGS, TDP-43/+ is elavGS, UAS-TDP-43(S)/+.elavGS, TDP-43/ATXN2-32Q is elavGS, UAS-TDP-43(S)/ UAS-ATXN2-32Q. elavGS, TDP-43/ΔPAM2 is elavGS, UAS-TDP-43(S)/ UAS-ATXN2-32Q.ΔPAM2. All flies raised with RU486 (20 μg/ml). *p<0.05, **p< 0.01, n.s. not significant. (ANOVA for significance, followed by Student’s t-test).