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. 2017 Jun 27;6:e24139. doi: 10.7554/eLife.24139

Figure 2. Poly(A) tail length determines cell-type and developmental stage-specific translation of PABPC1.

(A). Polysome profile of embryonic day 18 (E18) and adult mouse hearts. (B) Percentage of Pabpc1 and Gapdh mRNAs measured by qPCR in each fraction collected from the polysome profiling. (C) Neonatal and 8-week-old adult wild-type mice were pulsed with puromycin through an intraperitoneal injection. Forty-five minutes following injection, heart and liver tissues were harvested for immunoblotting with anti-puromycin antibody. De novo protein synthesis was quantified as the ratio of puromycin labeled peptides to total protein. (D) Fractional distribution of Pabpc1 mRNAs with short and long poly(A) tails in whole heart, C2C12 cells, cardiomyocytes (CMs), cardiac fibroblasts (CFs), whole heart after TAC surgery, and whole heart after exercise (measured by qPCR following poly(A) tail fractionation). (E) Poly(A) tail length status of Pabpc1 mRNA within P0 and adult heart RNP, monosome, and polysome fractions from sucrose gradients. (F) Pabpc1 single-molecule RNA-FISH in C2C12 myoblasts and myotubes. Data are mean ± s.d (n = 3); *p<0.05, **p<0.005 unpaired two-tailed t-test; NS, not significant.

DOI: http://dx.doi.org/10.7554/eLife.24139.006

Figure 2.

Figure 2—figure supplement 1. Regulation of PABPC1 expression in adult heart is independent of miRNAs or alternative splicing.

Figure 2—figure supplement 1.

Targeted deletion of Dicer in adult cardiomyocytes was obtained by treating 8-week-old Dicer f/f; MCM mice with tamoxifen (20 mg/Kg/day) for 5 consecutive days21. Forty-eight hours after the last injection, heart tissues were harvested. (A) Relative PABPC1 protein (immunoblots) or (B) mRNA (qPCR) levels were measured in the indicated samples. Fold change in PABPC1 protein was determined by relative quantification of band intensities, normalized to GAPDH (shown below the blots). Data are mean ± s.d (n = 3); unpaired two-tailed t-test. NS, not significant. (C) UCSC genome browser tracks of Pabpc1 mRNA in cardiomyocytes and fibroblasts from neonatal and adult mice16.
DOI: http://dx.doi.org/10.7554/eLife.24139.007

Figure 2—figure supplement 2. Limited influence for 5’ and 3’ untranslated regions (UTRs) of Pabpc1 on luciferase protein translation during C2C12 differentiation.

Figure 2—figure supplement 2.

(A) Representative immunoblot demonstrating a steady decrease in PABPC1 protein levels during C2C12 differentiation; p38 was used as a loading control. (B) Quantification of PABPC1 protein (immunoblots) and mRNA (qPCR) levels relative to p38 during C2C12 differentiation. (C) Schematic of the reporters designed to assess the effect of Pabpc1 5’ and 3’ UTRs on luciferase protein expression. (D) Relative luciferase activity derived from C2C12 cells transfected with different reporter constructs shows that only Pabpc1 5’ UTR has a moderate effect (twofold) on the reporter activity in differentiated myotubes, whereas the 3’ UTR and the combination of UTRs have no effects. Data are mean ± s.d (n = 3); *p<0.05, unpaired two-tailed t-test. NS, not significant.
DOI: http://dx.doi.org/10.7554/eLife.24139.008

Figure 2—figure supplement 3. Experimental design of northern blot and RNA isolation based on poly(A) tail length through gradient purification.

Figure 2—figure supplement 3.

(A) Oligo design for the Pabpc1 northern assay. Oligos were designed for the Pabpc1 mRNA to be targeted in an RNAseH digestion leaving 700nt downstream of the cleavage site and the native poly(A) tail. (B) Northern blot using a radiolabeled probe against Pabpc1 from E18 and 8-week-old adult mouse hearts following RNAseH cleavage. In the final lane, oligo dT was also added to the RNAseH reaction so that the Pabpc1 transcript was cleaved at both ends, leaving just the 700nt region. A similar RNAseH cleavage assay was used for GAPDH as a control. (C) Adult mouse heart total RNAs with and without RNAse T1 treatment were mixed with biotinylated oligo(dT) and bound to the streptavidin-conjugated beads. Different salt concentrations were used to elute mRNAs. The differing poly(A) tail lengths of eluted RNAs were checked by northern blot analysis using an oligo-dT40 probe. This served as optimizations for further purifications of short and long poly(A) tail RNAs that were subjected to qPCR analysis using gene-specific primers. (D) Poly(A) tail status of P0 and Adult Gapdh in polysome gradient fractions as a control experiment. Gapdh is enriched in the long-tailed and polysome fractions as expected.
DOI: http://dx.doi.org/10.7554/eLife.24139.009