Figure 5: Loss of TAF15 or FUS affects mRNA stability in human neural precursor cells. | Nature Communications

Figure 5: Loss of TAF15 or FUS affects mRNA stability in human neural precursor cells.

From: Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses

Figure 5

(a) Schematic of workflow. (1) Human iPSCs were differentiated into NPCs and stained for neuronal lineage markers DCX (left, green), NESTIN (right, red) and SOX2 (right, green) to confirm differentiation. 4,6-Diamidino-2-phenylindole (DAPI; blue) was used to locate nuclei. Scale bar, 25 μm. (2) NPCs were infected with virus expressing shRNAs against TAF15 or FUS and then treated with Actinomycin D for indicated duration. (3) Poly(A)-selected RNA was converted into libraries for sequencing, and sequencing reads were used to calculate mRNA half-lives. (b) Validation of shRNA-mediated knockdown of FUS or TAF15 in NPCs by western blot analysis. Representative western blot is shown from one replicate with quantifications from biological triplicate knockdown experiments. (c) For each gene, the coefficient of determination (R2) reflecting the fit of the RPKM values to a log-linear regression was computed. The cumulative distribution functions of the R2 values for all genes in the TAF15 depletion experiment are depicted for real and shuffled values. (d) Table displaying the number of mRNAs whose half-lives were destabilized, stabilized or unchanged by depletion of TAF15 or FUS. Half-life changes, measured as log2 (knockdown/control), that were greater than 1 were considered. (e) Heatmap of normalized RPKMs for stabilized and destabilized mRNAs upon shRNA-mediated knockdown of TAF15 or FUS. RPKMs are normalized for each gene to its RPKM at time 0. An asterisk indicates that the gene was examined for binding in f. (f) RNA immunoprecipitation was performed using antibodies against IgG (Control), TAF15 and FUS in NPCs. The relative fold change compared with the IgG control for genes that are stabilized by TAF15 loss was determined with qPCR. Values are means±s.d. for biological duplicates. Asterisk denotes a significant difference compared with IgG by Student’s t-test where **P<0.005 and *P<0.05. (g) RNA immunoprecipitation analysis as in f for mRNAs that were destabilized upon TAF15 loss.

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