The molecular basis of coupling between poly(A)-tail length and translational efficiency
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Evaluation Summary:
This manuscript addresses a long-standing question, namely how does the poly(A) tail influence translational efficiency? It will therefore be of broad interest to readers from many areas of molecular biology including those interested in translation, mRNA stability, development and gene expression in general. The authors convincingly set out three criteria that must be met for coupling of poly(A) tail length with translation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
In animal oocytes and early embryos, mRNA poly(A)-tail length strongly influences translational efficiency (TE), but later in development this coupling between tail length and TE disappears. Here, we elucidate how this coupling is first established and why it disappears. Overexpressing cytoplasmic poly(A)-binding protein (PABPC) in Xenopus oocytes specifically improved translation of short-tailed mRNAs, thereby diminishing coupling between tail length and TE. Thus, strong coupling requires limiting PABPC, implying that in coupled systems longer-tail mRNAs better compete for limiting PABPC. In addition to expressing excess PABPC, post-embryonic mammalian cell lines had two other properties that prevented strong coupling: terminal-uridylation-dependent destabilization of mRNAs lacking bound PABPC, and a regulatory regime wherein PABPC contributes minimally to TE. Thus, these results revealed three fundamental mechanistic requirements for coupling and defined the context-dependent functions for PABPC, which promotes TE but not mRNA stability in coupled systems and mRNA stability but not TE in uncoupled systems.
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Reviewer #3 (Public Review):
The manuscript by Xiang and Bartel explores the molecular coupling of poly(A) tail length and translational efficiency (TE) in frog oocytes and various mammalian cell lines. From their experiments they draw several broad conclusions. Firstly, it is that limiting amounts of PABPC in frog oocytes is the basis for coupling between poly(A) tail length and TE. Secondly, in mammalian somatic cell lines PABPC contributes little to TE and transcript with TUT4 and TUT7-mediated uridylation promoting degradation of transcript with short poly(A) tails. Overall, the experimental design is excellent. The conclusions drawn from the frog oocytes are strongly supported by the data provided whereas the cell line studies are more open to interpretation due to the drastic consequences of PABPC depletion.
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Reviewer #2 (Public Review):
Poly(A) tails are generally thought to stabilize mRNAs and promote translation. However, the mechanisms of this process have been difficult to experimentally assess due to the essential nature of poly(A) binding proteins, homeostatic mechanisms in gene expression, and the pleiotropic effects of altering the transcription, translation or mRNA decay machinery. The length of poly(A) tails are directly proportionally to translational efficiency in early development - the longer the tail, the more efficiently the mRNA is translated - possibly through a closed loop model. However, experiments in other cells, as well as in vitro reconstitution and imaging of single mRNAs in cells, do not support either coupling of poly(A) tail length and TE, or the closed loop model. Thus, it appears that there is a switch from …
Reviewer #2 (Public Review):
Poly(A) tails are generally thought to stabilize mRNAs and promote translation. However, the mechanisms of this process have been difficult to experimentally assess due to the essential nature of poly(A) binding proteins, homeostatic mechanisms in gene expression, and the pleiotropic effects of altering the transcription, translation or mRNA decay machinery. The length of poly(A) tails are directly proportionally to translational efficiency in early development - the longer the tail, the more efficiently the mRNA is translated - possibly through a closed loop model. However, experiments in other cells, as well as in vitro reconstitution and imaging of single mRNAs in cells, do not support either coupling of poly(A) tail length and TE, or the closed loop model. Thus, it appears that there is a switch from embryonic to post-embryonic regulation of TE. The mechanistic basis for this switch was unclear.
Here, Xiang and Bartel use reporter assays and transcriptome-wide sequencing technologies, alongside other complementary experiments, to determine the specific circumstances that permit coupling of poly(A) tail length and translational efficiency. The authors are able to synthesize many observations - both from their own lab and from others - to come up with a unified hypothesis. Many of the individual findings have been previously reported or hypothesized but no other work has brought all of these together in one study.
Overall, the data strongly support the conclusions. Importantly, several different cell types and systems are used. In addition, a number of different methods support the work - including reporter assays, global analyses, experiments in extracts, oocytes and cell lines, etc.
A description of events that lead to the switch from embryonic to post-embryonic regulation is still lacking. However, the insight provided here is substantial. It will have influence on many areas of study of gene expression - for example, it helps to explain discrepancies in miRNA function.
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Reviewer #1 (Public Review):
This is an excellent manuscript in which Bartel and colleagues use an abundance of approaches to provide compelling evidence relevant to the coupling between poly(A)-tail length and translational efficiency. Without reiterating the results, the data are convincing and the paper is clearly written. Any concerns are too trivial to articulate.
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Evaluation Summary:
This manuscript addresses a long-standing question, namely how does the poly(A) tail influence translational efficiency? It will therefore be of broad interest to readers from many areas of molecular biology including those interested in translation, mRNA stability, development and gene expression in general. The authors convincingly set out three criteria that must be met for coupling of poly(A) tail length with translation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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