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. 2016 Jun 22;5:e16030. doi: 10.7554/eLife.16030

Figure 4. Nkx2-5 functions together with Xrn2 to regulate APA.

(A) Xrn2 knockdown was analyzed by Western blotting. (B and C) qRT-PCR analysis of mRNAs expression of the long 3’ UTRs (B) and gene bodies (C) of Tnnt2 and Atp2a2 in Xrn2-knockdown eCMs, normalized to Rplp2. (D) Xrn2 expression levels were measured by qRT-PCR and normalized to Rplp2. (E) Xrn2 binding in eCMs transfected with the indicated siRNAs was analyzed by ChIP-qPCR. The control values were set to 1.0. (F) Long 3’ UTRs in eCMs transfected with the indicated siRNA were analyzed by Northern blotting using probes against Tnnt2 and Atp2a2 mRNA. Red brackets indicate mRNAs with long 3’ UTRs. (G) The original lengths of the Tnnt2 and Atp2a2 mRNAs and the lengths of the Tnnt2 and Atp2a2 mRNAs with long 3’ UTRs that were used in the Northern blot analysis were measured by BAS5000. The ratio of the siControl was set to 1.0. (H) Tnnt2 and Atp2a2 proteins in Nkx2-5 and Xrn2 knockdown eCMs were analyzed by western blotting. (I) The average profiles of the mRNAs with long 3’ UTRs that were increased in Nkx2-5-knockdown eCMs are shown in eCMs transfected with the indicated siRNAs. The gray area indicates the coding region. Significance was assessed using the two-sample Kolmogorov-Smirnov test. For B, C, D, E, and G, error bars indicate the mean ± s.e.m. (n = 3). *, p < 0.05.

DOI: http://dx.doi.org/10.7554/eLife.16030.021

Figure 4—source data 1. Source data for Figure 4 and Figure 4-figure supplement 1 and 2.
Numeric data for Figure 4B,C,D,E,G, Figure 4—figure supplement 1B, Figure 4—figure supplement 2.
DOI: http://dx.doi.org/10.7554/eLife.16030.022
elife-16030-fig4-data1.xlsx (40.8KB, xlsx)
DOI: 10.7554/eLife.16030.022

Figure 4.

Figure 4—figure supplement 1. Knockdowns of Nkx2-5 or Xrn2 affect the expression of the long 3’UTR regions in chromatin-fractioned RNA.

Figure 4—figure supplement 1.

(A) Genome browser representation of strand-specific chromatin-fractioned RNA-seq tag counts from eCMs transfected with the indicated siRNAs. (B) qRT-PCR analysis of mRNA expression of long 3’ UTRs of Tnnt2 and Atp2a2, normalized to Rplp2 on chromatin-fractioned RNA. Error bars indicate the mean ± s.e.m. (n = 3). *, p < 0.05. The arrow heads show polyadenylation sites.
DOI: http://dx.doi.org/10.7554/eLife.16030.023
Figure 4—figure supplement 2. Knockdown of Nkx2-5 affects Xrn2-binding to Myl7.

Figure 4—figure supplement 2.

Xrn2 binding in eCMs transfected with the indicated siRNAs was analyzed by ChIP-qPCR. The control values were set to 1.0. Error bars indicate the mean ± s.e.m. (n = 3). *, p < 0.05.
DOI: http://dx.doi.org/10.7554/eLife.16030.024
Figure 4—figure supplement 3. Nkx2-5 functions together with Xrn2 to regulate APA.

Figure 4—figure supplement 3.

The EtBr staining gels and the blottings of b-actin as an internal control are shown, related to Figure 4F.
DOI: http://dx.doi.org/10.7554/eLife.16030.025
Figure 4—figure supplement 4. Correlation analysis of Nkx2-5 binding and long 3’UTR expression.

Figure 4—figure supplement 4.

(A) The average profiles of the mRNAs with long 3’ UTRs in genes that Nkx2-5 binds to TTS (3997 genes) in eCMs transfected with the indicated siRNAs. The gray area indicates the coding region. Significance was assessed using the two-sample Kolmogorov-Smirnov test. (B) The average profiles of Nkx2-5, Gata4, and Tbx5 binding in genes that their long 3’ UTRs were increased in Nkx2-5-knockdown eCMs. (C) Correlation plot of long 3’UTR expression between indicated knockdowns and Pearson correlation, related to Figure 4I. (D) Violin plots of fold change in long 3’UTR expression with box plots.
DOI: http://dx.doi.org/10.7554/eLife.16030.026