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

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3′-untranslated regions (3′ UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons.


Supplementary Figure 2: Examples of ARE-containing mRNAs significantly upregulated in differentiated P19 cells.
Changes in the expression levels of ARE-containing mRNAs in differentiating P19 cells plated for 3.5 days after the EB/RA induction step. Top, 3'UTR diagrams showing positions of pA sites, AREs and primers used for RT-qPCR analyses. Long black ticks, canonical AAUAAA and AUUAAA cleavage/polyadenylation (pA) hexamers occurring within 10-30 nt upstream of the 3' end in least one cDNA or EST clone (UCSC Genome Browser) or onenucleotide modifications of these hexamers used as pA sites in at least five cDNA/EST clones. Short black ticks, AAUAAA and AUUAAA pA hexamers not supported by available cDNA clones. Red ovals, UAUUUAU motifs occurring as a part of ≥12 nt consecutive AUnucleotide sequences with the number of individual UAUUUAU heptamers indicated inside the oval. Red ticks, UAUUUAU motifs present within <12 nt AU sequences. Bottom, RT-qPCR relative expression data obtained for undifferentiated P19 cultures and cultures differentiated for 3.5 days using corresponding F/R primer pairs. Gene expression levels in the undifferentiated cells are set to 1. Data are averaged from 3 experiments ±SD and compared by t-test.

Supplementary Figure 3: ARE-dependent mRNA destabilization is virtually inactive in the TubβIII-positive neuron-like population of differentiated P19 cells.
EB/RA-differentiated transgenic P19 cells introduced in Fig. 2c-d were stained with TubβIIIspecific (Tuj1) antibody and analysed by FACS. dTomato expression levels (histogram on the right) were examined in the TubβIII-positive populations selected as shown in the two histograms on the left. No significant difference in dTomato expression was detected between dTom-3'HuR-pA2mut and dTom-3'HuR-pA2mut/ΔARE Tuj1-positive cells (Wilcoxon ranksum test p=0.24).

Supplementary Figure 4: ARE-dependent pathway is virtually inactive in primary neurons but readily detectable in embryonic fibroblasts.
Left, Mouse embryonic fibroblasts or primary cortical neurons were magnetically transfected with dTom-3'HuR-pA2mut and dTom-3'HuR-pA2mut/ΔARE constructs and the dTomato protein expression was analysed by flow cytometry. Right, no significant expression difference between the two constructs was detected for neurons (Wilcoxon rank-sum test p=0.76), whereas MEFs (middle) expressed significantly larger amounts of dTomato from dTom-3'HuR-pA2mut/ΔARE as compared to its ARE-containing counterpart (Wilcoxon rank-sum test p=1.8×10 -180 ).

Supplementary Figure 5: Expression levels of TTP protein and its paralog BRF1 decrease during neurogenesis.
Mouse embryonic stem cells (ESCs), neural stem cells (NSCs) and neurons prepared from E15.5 embryonic cortices were analysed by immunoblotting with antibodies recognizing TTP/Zfp36 (top) or two of its paralogs, BRF1/Zfp36l1 and BRF2/Zfp36l2 (middle). β-tubulin (Tubβ) is used as a lane loading control (bottom). Note that TTP and BRF1 are downregulated during neurogenesis, while BRF2 is not detectable in any of the three cell types.

Supplementary Figure 6: miR-9 represses TTP expression by interacting with its 3'UTR-specific target site.
(a) HEK293T cells were co-transfected with RLuc-3'TTP-wt and miR-9 expression plasmid or the corresponding empty vector. Firefly luciferase plasmid pEM231 4 was included as a normalization control. Luciferase expression was assayed 24 hours post transfection using Dual-Glo kit (Promega) and the data were processed as recommended. Two samples were additionally transfected with a TTP 3'UTR miR-9 site-specific target protector (Qiagen) at the final concentration of 50 nM (+) or 100 nM (++). Note that the target protector partially rescues the repressive effect of miR-9. Expression in the RLuc-3'TTP-wt / miR-vector cotransfected sample is set to 1. Data are averaged from 3 experiments ±SD and compared by ttest. (b) EB/RA-treated P19 cells were transfected with 125 nM of TTP 3'UTR miR-9 sitespecific target protector and the expression of TTP protein was analysed by immunoblotting 48 hours post transfection. β-tubulin (Tubβ) is used as a lane loading control.

Supplementary Figure 7: TTP down-regulation is necessary for proper neuronal differentiation of EB/RA-induced P19 cells.
(a) RT-qPCR analyses of EB/RA-differentiated P19-TTP cells described in Fig. 4. Note that transgenic TTP protein induced by Dox diminishes expression of neuronal marker genes either lacking (Map2 and L1cam) or containing a single UAUUUAU motif in their 3'UTRs (Syp and Syt4), while having no significant effect on these genes in control P19 cells. Data are averaged from 3 amplifications ±SD and compared by t-test. (b) Immunoblot analysis of EB/RA-differentiated P19-TTP cells showing reduced expression of Map2 protein in Doxinduced P19-TTP cells as compared to the corresponding Dox(-) control. Antibody against βtubulin (Tubβ) is used to control lane loading.