Hundreds of thousands of different proteins are needed to build and maintain the cells in the human body. All proteins are produced when copies of genetic information in the form of molecules of messenger RNA (mRNAs) are translated by the ribosome. The rate at which proteins are made varies widely between different tissues and at different times. In particular, the adult heart has one of the lowest rates of protein production, though this rate can increase markedly during exercise and heart disease. The mechanisms that drive this kind of dynamic change in protein production remain unclear. A better understanding of this process would tell scientists more about how and why cells regulate the translation of mRNAs in general, and might uncover new ways for treating heart disease.

Molecules of mRNA are composed of smaller building blocks called nucleotides. All mature mRNAs in humans have a long stretch at one end that contains the nucleotide adenosine – commonly referred to as A for short – repeated 200 to 300 times. This sequence is called the poly(A) tail, and specific proteins can bind to this tail and determine the final fate of the mRNA, such as how efficiently it is translated. One such poly(A) binding protein, called PABPC1, is known to promote mRNA translation.

Now, Chorghade, Seimetz et al. examine how PABPC1 regulates protein production in mice and human cells. The experiments reveal that, before birth, ample amounts of PABPC1 are found in the heart, but that this protein is undetectable in the hearts of adults. Further experiments showed that the levels of the mRNA for PABPC1 in the heart are the same before birth and in adulthood. So why is the mRNA for PABPC1 translated inefficiently in adult hearts? In general, mRNAs with long tails tend to be translated more efficiently compared to those with short tails, and it turns out that the mRNA for PABPC1 has a substantially shorter poly(A) tail in the adult heart. This tail shortening limits the translation of this specific mRNA, which leads to reduced protein production.

Chorghade, Seimetz et al. also showed that the length of the poly(A) tail on the mRNA and the levels of the PABPC1 protein are restored in adult hearts during a condition known as hypertrophy. This state of hypertrophy can be triggered by exercise or heart disease and is marked by an increase in the size of individual heart cells and enhanced protein production. Finally, Chorghade, Seimetz et al. found that experimentally raising the levels of PABPC1 in adult hearts could, by itself, make the heart cells produce more protein and the heart grow more. Further studies will explore if other mRNAs in the heart also undergo similar changes and whether this is a general mechanism for controlling protein production.

Cellular growth and function depend heavily on protein synthesis, which is often considered a constitutive activity for a cell. However, it is becoming clear that global protein synthesis rates are not always static, that they vary widely among cell types, and that these differences are necessary for normal tissue development and homeostasis (Buszczak et al., 2014). Particularly, the rate of protein synthesis in adult heart is one of the lowest amongst different tissues but increases markedly in response to stress and hypertrophic stimuli (Garlick et al., 1980; Lewis et al., 1984). The molecular basis for these historical observations, however, is still poorly understood.

Translation initiation is the rate-limiting step in protein synthesis (Aitken and Lorsch, 2012; Hinnebusch et al., 2016; Sonenberg and Hinnebusch, 2009). Interactions between the 5’ m⁷GpppN cap structure, the pre-initiation factors (including eIF4A, eIF4E, and eIF4G), and poly(A)-binding protein C1 (PABPC1) form a stable, looped mRNP complex (Amrani et al., 2008; Gallie, 1991; Park et al., 2011; Safaee et al., 2012; Tarun and Sachs, 1996; Wells et al., 1998) that stimulates translation while safeguarding the mRNA from exonucleases (Coller et al., 1998; Gray et al., 2000; Kahvejian et al., 2005; Lewis et al., 2017; Zekri et al., 2013). Based on these central roles, PABPC1 is thought to be ubiquitously expressed and serve ‘house-keeping’ roles in protein synthesis.

Here, we report that PABPC1 protein expression is post-transcriptionally silenced in adult human and mouse hearts through shortening of its mRNA poly(A) tail, which results in reduced polysome association and translation of Pabpc1 transcripts. The developmental silencing of PABPC1 is cardiomyocyte-specific and reversible. We show that Pabpc1 poly(A) tail length and protein expression are restored during adult-onset cardiac hypertrophy stimulated by endurance exercise or heart disease. Furthermore, we demonstrate that PABPC1 re-expression and its interaction with eIF4G are necessary and sufficient to globally stimulate translation and physiologic growth of cardiomyocytes. These findings reveal a novel, poly(A) tail-based regulatory mechanism in the heart that dynamically controls PABPC1 expression and subsequent protein synthesis in response to developmental and hypertrophic signals.

The association of eIF4F complex with the 5’ m⁷G cap structure is stabilized through eIF4G-PABPC1 interactions, which promote ribosomal recruitment and translation initiation (Amrani et al., 2008; Gallie, 1991; Safaee et al., 2012; Tarun and Sachs, 1996; Wells et al., 1998). We have discovered that PABPC1 protein levels in the adult mouse heart are drastically lower relative to the embryonic day (E)17 stage (Figure 1A,B). Parallel examination of Pabpc1 mRNA abundance unexpectedly showed only a modest decrease after birth (Figure 1B). A similarly striking reduction in PABPC1 protein, but not mRNA levels, was observed in adult versus fetal human hearts indicating PABPC1 silencing is post-transcriptional and evolutionarily conserved (Figure 1C,D). We inspected PABPC1 mRNA and protein abundance in several other mouse and human tissues and determined that the postnatal silencing of PABPC1 is muscle-specific (Figure 1A and Figure 1—figure supplement 1A–D). Coimmunofluorescent staining of PABPC1 with a cardiomyocyte marker (TNNT2) combined with immunoblot analyses of purified cell types revealed that while PABPC1 is abundantly expressed in all cells within the neonatal heart, it is selectively silenced in adult cardiomyocytes (Figure 1E,J and Figure 1—figure supplements 1E and 2).

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We sought to determine whether PABPC1 protein is re-expressed in the adult heart during hypertrophy, a condition accompanied by increased size of individual cardiomyocytes, enhanced protein synthesis, and induction of many fetal genes (Hill and Olson, 2008; Maillet et al., 2013; Towbin and Bowles, 2002). We used thoracic aortic constriction (TAC) and endurance exercise training in mice as models of pathologic and physiologic hypertrophy, respectively (De Lisio and Parise, 2012; Kalsotra et al., 2014). PABPC1 protein levels were markedly induced under both conditions without any change in mRNA abundance (Figure 1K,L). These results illustrate that post-transcriptional silencing of PABPC1 in the adult heart is reversed during cardiac hypertrophy.

To probe whether the disappearance of PABPC1 protein in the adult heart is due to a reduction of synthesis, we conducted polysome profiling of E18 and adult mouse hearts (Figure 2A). Quantitative PCR (qPCR) analyses of fractionated lysates showed a significant shift of Pabpc1 mRNAs away from polyribosomes to the mRNP/monosome fractions in the adult hearts, demonstrating reduced accessibility to the translational machinery in contrast to the E18 hearts. Control Gapdh mRNA remained associated with polyribosomes at both developmental stages (Figure 2B).

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Intriguingly, in comparison to E18, the adult heart polysome profiles exhibited a decrease in the number of polyribosomes with a corresponding increase of 80S monosomes, which reflects reduced translation efficiency (Figure 2A). To further quantify global protein synthesis rates in vivo, we pulse-labeled wild-type neonates and adult mice with puromycin followed by immunoblotting using an anti-puromycin antibody (i.e. SUnSET assays) (Goodman et al., 2011; Schmidt et al., 2009). We detected a robust decrease in puromycin-labeled peptides in adult versus neonatal hearts but not in liver tissues (Figure 2C). Lower protein synthetic capacity of adult striated muscle compared to other tissues was first observed more than three decades ago (Garlick et al., 1980; Lewis et al., 1984). Our data showing muscle-specific silencing of PABPC1 thus offers a plausible molecular basis for these historical observations. Low-level protein synthesis in the absence of PABPC1 could presumably result from alternative mRNA circularization mechanisms (Bukhari et al., 2016; Lin et al., 2016; Wang et al., 2015) or translation of some mRNAs in a closed loop-independent manner (Archer et al., 2015; Costello et al., 2015).

Next, we investigated the molecular mechanisms responsible for inefficient translation of PABPC1. It appears that suppressed PABPC1 translation in the adult heart is microRNA-independent, as we saw no change in PABPC1 protein or mRNA in tamoxifen-inducible, heart-specific adult dicer knockouts (Figure 2—figure supplement 1A,B), which are defective in microRNA processing (Kalsotra et al., 2010). We analyzed available RNA-sequencing data from neonatal and adult mouse cardiomyocytes and fibroblasts (Giudice et al., 2014) and found no evidence for alternative splicing or a developmental change in Pabpc1 5’- or 3’- untranslated regions (UTRs) in either cell types (Figure 2—figure supplement 1C). We reasoned that trans-acting factor(s) might bind to the sequence elements within Pabpc1 UTRs to suppress its translation. To test this hypothesis, we constructed luciferase reporters fused to 5’-, 3’- or both mouse Pabpc1 UTRs and transfected them into C2C12 myoblasts. C2C12 cells exhibit marked downregulation of endogenous PABPC1 protein (~10 fold) but not mRNA levels when differentiated into myotubes (Figure 2—figure supplement 2A,B). Amongst the different reporters, only Pabpc1 5’-UTR showed a modest (<2-fold) decrease in luciferase (Rluc/Fluc) activity during myoblast-to-myotube differentiation (Figure 2—figure supplement 2C,D). Although PABPC1 binding to an A-rich element within its 5′-UTR could auto-regulate its translation (de Melo Neto et al., 1995; Kini et al., 2016), the relatively mild effects of 5’-UTR in reporter assays along with decreasing PABPC1 protein levels during muscle development argue against auto-regulatory feedback as the primary mechanism inhibiting Pabpc1 translation.

Besides UTRs, poly(A) tail length is another major determinant for protein synthesis; mRNAs containing longer tails being more stable and translated more efficiently (Eichhorn et al., 2016; Lim et al., 2016; Weill et al., 2012). Therefore, we tested whether Pabpc1 mRNAs are deadenylated in the adult heart. Both RNaseH cleavage assay and qPCR of long- and short-tailed mRNAs (fractionated by affinity chromatography) demonstrated significant Pabpc1 deadenylation in adult versus E18 hearts and in myotubes versus myoblasts (Figure 2C and Figure 2—figure supplement 3A–C). Pabpc1 poly(A) tail length in E18 hearts was estimated to be ~150 nucleotides (nts), whereas it was reduced to ~20 nts in adult hearts (Figure 2—figure supplement 3B). Gapdh poly(A) tail length was measured as a control and showed no difference between E18 and adult hearts. Importantly, shortening of Pabpc1 poly(A) tail in the adult heart was cardiomyocyte-specific; and partly reversed after TAC or endurance exercise (Figure 2C). In addition, Pabpc1 poly(A) tail length was strongly correlated to its association with monosomes and polysomes. At P0, Pabpc1 mRNAs were almost exclusively long-tailed and in polysomes, whereas adult Pabpc1 fractionated majorly into short-tailed and monosome fractions (Figure 2E). Gapdh poly(A) tail length remained unchanged and primarily associated with polysomes at both developmental stages (Figure 2—figure supplement 3D). We further explored if inhibition of Pabpc1 translation could be due to nuclear retention of Pabpc1 transcripts. Single-molecule RNA FISH, however, showed primarily cytoplasmic staining without any noticeable difference in Pabpc1 mRNA localization between myoblasts and myotubes indicating poly(A) tail status does not impact Pabpc1 nucleo-cytoplasmic export in these cells (Figure 2F). Together, these results provide compelling evidence that Pabpc1 poly(A) tail length is dynamically regulated during cardiac development and hypertrophy, and that poly(A) tail shortening limits Pabpc1 mRNA translation in the adult heart.

Because PABPC1 is upregulated in hypertrophy, we tested whether it is required for stimulus-induced growth of cardiomyocytes. PABPC1-depleted neonatal mouse cardiomyocytes were viable but resistant to isoproterenol (Iso) or triiodothyronine (T3)-induced hypertrophy (Figure 3A–I). Metabolic labeling of cardiomyocytes with an alkyne-modified glycine analog, L-homopropargylglycine (HPG), to measure newly synthesized proteins revealed that PABPC1 knockdown inhibited the normal surge in protein synthesis rate evoked by Iso or T3 stimulation (Figure 3J,K). Furthermore, we found that PABPC1 deficiency blocked protein but not mRNA upregulation of the hypertrophic markers Acta1, Myh7 and Anp (Figure 3L,M) indicating that while new protein synthesis is impaired, the transcriptional response to hypertrophic stimuli is still functional in these cells (Kim et al., 2008; Sergeeva and Christoffels, 2013). These results demonstrate that PABPC1 depletion does not affect the transcriptional regulatory circuits or mRNA stability of these hypertrophy markers.

Figure 3

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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

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PABPC1 contains four RNA recognition motifs (RRM1-4), a linker region and a C-terminal MLLE domain (Gray et al., 2000). While all four RRMs are capable of binding to poly(A) RNA, RRM2 preferentially interacts with eIF4G to stimulate cap-dependent translation (Safaee et al., 2012). Hence, we investigated if PABPC1 interactions with eIF4G are necessary for triggering cardiomyocyte hypertrophy and new protein synthesis. The M161A mutation was previously shown to disrupt PABPC1 interactions with eIF4G (Kahvejian et al., 2005). However, we decided to use the recently available structural information (Safaee et al., 2012) to engineer additional mutations within the RRM2 domain of PABPC1 to completely abolish its interactions with eIF4G (Figure 4—figure supplement 1A). We confirmed that although PABPC1_mRRM2 fails to interact with eIF4G1 in coimmunoprecipitation and in vitro binding assays, it binds to poly(A) RNA with similar affinities as wild-type PABPC1 (Figure 4—figure supplement 1B–F).

Next, we carried out rescue experiments wherein endogenous PABPC1 in cardiomyocytes was silenced using a 3’-UTR-targeted siRNA followed by adenoviral transduction of GFP, siRNA-resistant wild type, or PABPC1_mRRM2 cDNAs (Figure 4—figure supplement 1G). Supplementing PABPC1-deficient cardiomyocytes with wild type PABPC1 re-sensitized the hypertrophic growth response to Iso (Figure 4A–M), reversed the block in new protein synthesis (Figure 4N–P) and restored translation of hypertrophic markers in these cells (Figure 4Q). PABPC1_mRRM2, however, failed to rescue any of these phenotypes (Figure 4A–Q) underscoring that PABPC1–eIF4G interactions are essential to evoke cardiomyocyte hypertrophy and stimulate translation in response to hypertrophic signals.

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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

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To further determine if PABPC1 upregulation is sufficient to drive cardiac hypertrophy, we generated a doxycycline-inducible, cardiomyocyte-specific PABPC1 transgenic mouse model (Figure 5 and Figure 5—figure supplement 1A,B). Ectopic expression of FLAG-tagged PABPC1 protein in adult mouse hearts showed the expected cytoplasmic distribution (Figure 5E,F) and was estimated approximately 12-fold higher over endogenous levels (Figure 5—figure supplement 1C,D). Remarkably, forced PABPC1 expression in the adult myocardium led to increased atrial and ventricular size (Figure 5A–D), a significant elevation in heart-to-body weight ratios, and larger cardiomyocyte cross-sectional areas compared to uninduced littermate controls (Figure 5G–J). We also observed significant increase in global protein synthesis rates along with upregulation of many physiological but not pathological hypertrophic markers in PABPC1 transgenic hearts (Figure 5K,L). Notably, PABPC1 transgenic mice did not exhibit premature lethality or cardiac dilation even when induced for 6 months. Moreover, long-term PABPC1 induction did not cause a drop in performance in treadmill tests, deterioration in cardiac contractility, or deficits in systolic and diastolic function (Figure 5—figure supplement 1E,F). Also, histologically, we did not observe any myofiber disarray or fibrosis (Figure 5 and Figure 5—figure supplement 2) suggesting hypertrophy in these animals is compensated and does not progress to heart failure, but rather mimics the physiologic form.

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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

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In conclusion, we have uncovered that poly(A) tail length shortening suppresses Pabpc1 mRNA translation in mature cardiomyocytes, thereby reducing overall protein synthesis rates in the adult heart. Intriguingly, despite having a short poly(A) tail, Pabpc1 transcripts remain stable and are not completely deadenylated or degraded. This might be due to binding of trans-acting factor(s), the presence of RNA structural element(s), or RNA modifications that act to stabilize Pabpc1 in adult cardiomyocytes (Lewis et al., 2017). We further demonstrate that PABPC1 protein expression and poly(A) tail length are partially restored during adult-onset cardiac hypertrophy to activate new protein synthesis and physiologic growth of cardiomyocytes. PABPC1 was also shown to be both necessary and sufficient to drive cardiomyocyte hypertrophy. Thus, dynamic control of PABPC1 serves an adaptive role in stimulating a beneficial form of cardiac hypertrophy that may be physiologically advantageous to the failing or dilated myocardium.

Two genome-wide studies found a weak correlation between poly(A) tail lengths and translational efficiency of mRNAs in cell culture (Chang et al., 2014; Subtelny et al., 2014). While Subtelny et al. observed clear coupling between poly(A) tail length and translation at early developmental stages in Xenopus and zebra fish, the association became less apparent in the adults. Thus, it is plausible that at steady states, poly(A) tail length is less critical for translation but becomes a determinant in certain tissue contexts, developmental stages, cell cycle regulation, daily rhythmic oscillations of protein synthesis, or cellular stress (Besse and Ephrussi, 2008; Kojima et al., 2012; Park et al., 2016). Furthermore, stronger relationships between poly(A) tail length and mRNA translatability may emerge under conditions where PABPC1 protein concentrations are limiting; our data from adult cardiomyocytes supports this hypothesis.

Taken together, our findings not only provide insight into the long-standing question of how the heart regulates protein synthesis during development and hypertrophy but also provide a new direction to explore therapeutic interventions. These results set the stage for elucidating the signaling cascades that regulate Pabpc1 poly(A) tail length in cardiomyocytes, determining mechanistically how those dynamics are achieved, and probing the general roles for poly(A) tail length in translation control within the heart and other somatic tissues.

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Author details

1. Sandip Chorghade

   Department of Biochemistry, University of Illinois, Illinois, United States

   Contribution

   SC, Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft

   Contributed equally with

   Joseph Seimetz

   Competing interests

   The authors declare that no competing interests exist.

2. Joseph Seimetz

   Department of Biochemistry, University of Illinois, Illinois, United States

   Contribution

   JS, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Contributed equally with

   Sandip Chorghade

   Competing interests

   The authors declare that no competing interests exist.

3. Russell Emmons

   Department of Kinesiology and Community Health, University of Illinois, Illinois, United States

   Contribution

   RE, Data curation, Formal analysis

   Competing interests

   The authors declare that no competing interests exist.

4. Jing Yang

   Department of Comparative Biosciences, University of Illinois, Illinois, United States

   Contribution

   JY, Resources, Investigation

   Competing interests

   The authors declare that no competing interests exist.

5. Stefan M Bresson

   Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   SMB, Data curation, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

6. Michael De Lisio

   1. Department of Kinesiology and Community Health, University of Illinois, Illinois, United States
   2. School of Human Kinetics, University of Ottawa, Ottawa, Canada

   Contribution

   MDL, Resources, Formal analysis, Investigation

   Competing interests

   The authors declare that no competing interests exist.

7. Gianni Parise

   Department of Kinesiology, McMaster University, Hamilton, Canada

   Contribution

   GP, Resources

   Competing interests

   The authors declare that no competing interests exist.

8. Nicholas K Conrad

   Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   NKC, Conceptualization, Formal analysis, Supervision, Investigation

   Competing interests

   The authors declare that no competing interests exist.

9. Auinash Kalsotra

   1. Department of Biochemistry, University of Illinois, Illinois, United States
   2. Carl R. Woese Institute of Genomic Biology, University of Illinois, Illinois, United States

   Contribution

   AK, Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration, Writing—review and editing

   For correspondence

   kalsotra@illinois.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-1011-0006

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