Poly(A) tail length regulates PABPC1 expression to tune translation in the heart

Essential revisions:

1) The authors state that they uncovered a poly(A)-based regulatory mechanism. There is a change in poly(A) tail length of PABPC1 mRNA, but data supporting a mechanistic role for this is missing. The questions that need to be answered are: What is the poly(A) tail status of the reporter mRNAs in C2C12 cells?

We appreciate the reviewer’s suggestion to measure poly(A) tail lengths of Pabpc1 reporter mRNAs. This is an interesting experiment, but somewhat artificial because our reporters contain strong heterologous polyadenylation elements as part of the plasmid backbone. Instead, we measured the poly(A) tail status of endogenous Pabpc1 mRNAs in C2C12 cells. The poly(A) tail length data obtained were consistent with reduced PABPC1 protein levels seen during myoblast-to-myotube differentiation (Figure 2—figure supplement 2A,B) such that Pabpc1 mRNAs were significantly deadenylated in myotubes when compared to myoblasts. These new data are added to the revised Figure 2D.

What is the poly(A) status of PABPC1 mRNA in the different fractions across the gradient in E18 and adult heart? Is there a correlation between polysome association and poly(A) tail length, does poly(A) tail length drive polysome association or does it protect/enhance deadenylation? It needs to be clear whether changes in poly(A) tail length are a cause, effect or bystander.

We have addressed this concern of the reviewer to provide further evidence for the correlation of Pabpc1 poly(A) tail length and polysome association. An additional experiment measuring Pabpc1 mRNA levels in short and long poly(A) tail fractions from polysome gradients has been added. In the neonatal heart, Pabpc1 has long poly(A) tail length and associates exclusively with polysomes, whereas in the adult heart most Pabpc1 mRNAs are short-tailed and associate with monosome and RNP fractions (Figure 2E). Poly(A) tail length of Gapdh mRNAs remain unchanged and primarily associate with polysomes at both developmental stages (Figure 2—figure supplement 3D). Thus, these results point towards a causal effect of poly(A) shortening in suppressing Pabpc1 translation in the adult heart.

The experiment in RRL is not informative and should be deleted.

As requested, we have removed the RRL in vitro translation data from the revised Figure 2.

2) Polysome profiles should be provided for the PABPC1 overexpression in mouse heart experiment to establish that this does indeed result in a change in translational utilization of mRNAs as posited.

We appreciate the reviewer’s suggestion to demonstrate the effect of PABPC1 re-expression on translation. However, we feel that polysome profiles are, by nature, qualitative and believe that our in vivo experiment utilizing the SUnSET method in PABPC1 re-expressing mice (Figure 5K) is not only quantitative, but also more informative regarding the impact of PABPC1 on global translation in the heart. The SUnSET assay has become widely accepted in the field to measure protein synthesis and has been previously demonstrated to match polysome traces (Genes Dev. (2016) 30:1-6; Nat Commun (2016) 11776; FASEB J (2017) 30; 2: 798-812).

3) Delete Figure 1—figure supplement 1C – [or provide validation for the antibody].

Anti-PABPC1 antibody (Abcam ab21060) used in the Human protein atlas database (see http://www.proteinatlas.org/ENSG00000070756-PABPC1/antibody) has been previously validated to specifically cross-react with human PABPC1. siRNA-based validation of this antibody is available in J Virol. 2013 Jan;87(1):243-56.

Figure 2—figure supplement 3B – PABPC1 mRNA levels should not be plotted on a log scale – which masks any changes between fetal and adult heart.

Thank you for raising this point. We have revised the Figure 2—figure supplement 2B by plotting the PABPC1 mRNA and protein level data on a normal scale in the graph.

For Figure 1E-J -please provide single channel images in main/supplemental figures.

As requested, we have now provided the single channel images in the Figure 1—figure supplement 2.

Clarify how protein levels were quantified (e.g. in WB- digital acquisition system).

We have expanded the description of methods used to quantify the protein levels. Briefly, the blots were treated with an ECL substrate, visualized on a ChemiDoc digital acquisition system, and quantified using Image Lab Software.

Some stats are missing in places e.g. Figure 1D,B, Figure 2—figure supplement 3B.

We thank the reviewers for pointing out this oversight. We have now added the statistics, p values and n numbers to Figure 1B and Figure 2—figure supplement 2B. The western blot data in Figure 1D, is derived from a single pool of fetal and adult human heart protein samples and this information is provided under the Methods section.

4) Figure 2: If the model of poly(A) tail length regulation is correct, then one would expect that polysome-associated Pabpc1 mRNA would increase under stress conditions. The addition of these experimental data would bolster the overall conclusion of the study.

We understand the reviewer’s curiosity about the increase in polysome association of Pabpc1 mRNA under stress conditions. These lines of investigations are logical extensions of our work, which we will be entertaining and testing in the future but they fall outside the scope of this current study.

5) Results and Discussion, seventh paragraph, Figure 3 and Figure S3—figure supplement 2A. Although PABPC1 plays an important role in mRNA stability in addition to translation, there are increased levels of Acta1, Myh7 and Anp mRNAs in PabpC1-knockown (KD) cells. Why are these mRNAs so stable in the absence of PABPC1?

We thank the reviewers for raising this important point. As has been previously reported, we believe that increased mRNA abundance of Acta1, Myh7 and Anp in cardiomyocytes after Isoproterenol or T3 stimulation is due to increased transcription of these hypertrophy markers (J Clin Invest. (2008) 118(1): 124-132; Biochim Biophys Acta. (2013) 1832 (12): 2403-13). However, as the reviewer pointed out, it is interesting to note that the increased mRNA abundance of Acta1, Myh7 and Anp is maintained even in PABPC1 depleted cells after Iso or T3 stimulation. These results imply that PABPC1 knockdown does not (i) interfere with the transcriptional regulatory circuits; and/or (ii) have a major impact on the mRNA stability of these genes. We have added this discussion to the revised manuscript.

6) Results and Discussion, in the conclusion, the authors need to address the question as to why is the Pabpc1 mRNA harboring a short poly(A) tail stable in mature cardiomyocytes in the absence of PABPC1?

Thank you for this comment. It is indeed interesting that in spite of harboring short poly(A) tails, Pabpc1 mRNA is stable in mature cardiomyocytes. This stabilization could be imparted through binding of trans-acting factor(s), due to the presence of RNA structural element(s), or RNA modifications that may inhibit further recruitment/activity of deadenylases. We have mentioned these possibilities now in the Discussion section.