Figure 2: Activity of the TTP/ARE pathway is reduced in cells undergoing neural differentiation. | Nature Communications

Figure 2: Activity of the TTP/ARE pathway is reduced in cells undergoing neural differentiation.

From: A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status

Figure 2

(a) CLIP assay diagram (see Methods for details). (b) RT–qPCR analysis of mRNA ultraviolet-crosslinked and co-immunoprecipitated with a TTP-specific rabbit antibody from undifferentiated P19 cells. Note that the anti-TTP antibody co-immunoprecipitates significantly larger fractions of ARE-containing mRNA species (HuR, Tubb3, HuB, HuC and Nova1) than corresponding unspecific rabbit IgG control (set to 1). On the other hand, no significant difference between anti-TTP and the control is detected for the Alas1 mRNA encoding aminolevulinic acid synthase 1 and containing no UAUUUAU motifs. Data are averaged from three experiments±s.d. and normalized to background crosslinking signal from Gusb ‘housekeeping’ mRNA lacking UAUUUAU’s. (c) dTomato transgenes fused with PAS2-mutated HuR 3′ UTRs either containing the WT ARE (dTom-3′HuR-pA2mut) or lacking this sequence (dTom-3′HuR-pA2mut/ΔARE). (d) Expression levels of the dTomato transgenes introduced in c were measured using flow cytometry. Note that expression of dTom-3′HuR-pA2mut/ΔARE is significantly higher than that of dTom-3′HuR-pA2mut in undifferentiated P19 but not in EB/RA-differentiated P19 cells. Background fluorescence of unmodified P19-A9 cells is plotted for reference.

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