Poly(A) tail length regulates PABPC1 expression to tune translation in the heart
- University of Illinois, United States
- University of Texas Southwestern Medical Center, United States
- University of Ottawa, Canada
- McMaster University, Canada
Figures
Figure 1 with 2 supplements
PABPC1 is dynamically regulated during cardiac development and hypertrophy.
(A–D) Relative quantification of PABPC1 protein (immunoblots) and mRNA (qPCR) levels normalized to GAPDH during mouse heart and liver development (A, B) and in human fetal and adult hearts (C, D). (E…
Figure 1—figure supplement 1
Post-transcriptional silencing of PABPC1 is muscle-specific.
(A) Immunoblots for PABPC1 from postnatal day 0 (P0) and 8-week-old adult mice from the indicated tissue with GAPDH as a loading control. (B) Quantification of PABPC1 protein and mRNA expression …
Figure 1—figure supplement 2
Post-transcriptional silencing of PABPC1 is muscle-specific.
Single-channel immunofluorescent images of mouse postnatal day 0 (P0) and 8-week-old adult hearts stained for PABPC1 (red), cardiac troponinT (green), and DAPI (blue).
Figure 2 with 3 supplements
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 …
Figure 2—figure supplement 1
Regulation of PABPC1 expression in adult heart is independent of miRNAs or alternative splicing.
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 …
Figure 2—figure supplement 2
Limited influence for 5’ and 3’ untranslated regions (UTRs) of Pabpc1 on luciferase protein translation during C2C12 differentiation.
(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 …
Figure 2—figure supplement 3
Experimental design of northern blot and RNA isolation based on poly(A) tail length through gradient purification.
(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) …
Figure 3
Knockdown of PABPC1 in neonatal mouse cardiomyocytes prevents stimulus-induced hypertrophy and protein synthesis.
(A–F) Primary cardiomyocytes isolated from newborn mice were transfected with siRNA against control Luciferase or Pabpc1. Twelve hours following transfection, cells were treated with isoproterenol …
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Figure 3—source data 1
Source data for cell area of cultured neonatal cardiomyocytes treated with siRNA and either Iso or T3.
- https://doi.org/10.7554/eLife.24139.011
Figure 4 with 1 supplement
PABPC1–eIF4G1 interactions control stimulus-induced new protein synthesis and hypertrophy.
(A–L) Representative images of neonatal cardiomyocytes infected with adenovirus expressing GFP, wildtype PABPC1, or a PABPC1 RRM2 mutant (that does not interact with eIF4G1), transfected with siRNA …
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Figure 4—source data 1
Source data for cell area of cultured neonatal cardiomyocytes after treatment with siRNA, adenovirus, and Iso.
- https://doi.org/10.7554/eLife.24139.013
Figure 4—figure supplement 1
PABPC1mRRM2 can bind to poly(A) RNA but does not interact with eIF4G1.
(A) Schematic of the PABPC1 protein with key domains labeled. Mutations of the RRM2 domain (PABPC1mRRM2) with eight amino acid replacements to interrupt PABPC1-eIF4G binding were made using the …
Figure 5 with 2 supplements
Forced expression of PABPC1 in adult cardiomyocytes induces physiologic hypertrophy.
(A–H) Representative whole heart, H&E, immunofluorescent, and WGA-stained sections of 2-week doxycycline (Dox)-induced MHCrtTA transgenic controls and TRE-PABPC1; MHCrtTA bitransgenic mice. (I) …
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Figure 5—source data 1
Source data for cardiomyocyte areas performed on WGA stained heart tissue sections of 2-week doxycycline-induced MHCrtTA transgenic controls and TRE-PABPC1; MHCrtTA bitransgenic mice.
- https://doi.org/10.7554/eLife.24139.016
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Figure 5—source data 2
Source data for Figure 5—figure supplement 1.
Sheet 1: Source data for exercise performance between 6-month doxycycline induced MHCrtTA transgenic controls and TRE-PABPC1; MHCrtTA bitransgenic mice. Sheet 2: Source data for cardiac function tests between 6-month doxycycline-induced MHCrtTA transgenic controls and TRE-PABPC1; MHCrtTA bitransgenic mice.
- https://doi.org/10.7554/eLife.24139.017
Figure 5—figure supplement 1
Generation of tetracycline-inducible, heart-specific PABPC1 transgenic mouse model.
(A) The TRE-PABPC1 construct expresses mouse PABPC1 containing an N-terminal Flag tag driven by a TRE and a CMV minimal promoter. TRE-PABPC1 mice were mated with Myh6-rtTA (MHCrtTA) mice to generate …
Figure 5—figure supplement 2
Induced expression of cardiomyocyte specific PABPC1 does not lead to damage.
Histological sections of 6-month 2 g/kg doxycycline (Dox)-induced MHCrtTA transgenic and TRE-PABPC1; MHCrtTA bitransgenic mice. Hematoxylin and eosin or trichrome staining shows normal …
Additional files
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Supplementary file 1
This document contains detailed information on tools used in the study. Sheet 1 contains the forward and reverse sequences of all primers organized by RT-qPCR, genotyping of mouse lines, cloning for plasmid constructs, RNAse H cleavage, and generation of northern blot probes. Sheet 2 contains antigen, manufacturer, product number, and RRID information for all antibodies used. Sheet 3 contains the probe sequences for Pabpc1 RNA-FISH.
- https://doi.org/10.7554/eLife.24139.020
