Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation through mRNA lifespan with most impact on short tails
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
Abstract
La-related protein 4 (LARP4) directly binds both poly(A) and poly(A)-binding protein (PABP). LARP4 was shown to promote poly(A) tail (PAT) lengthening and stabilization of individual mRNAs presumably by protection from deadenylation (Mattijssen et al., 2017). We developed a nucleotide resolution transcriptome-wide, single molecule SM-PAT-seq method. This revealed LARP4 effects on a wide range of PAT lengths for human mRNAs and mouse mRNAs from LARP4 knockout (KO) and control cells. LARP4 effects are clear on long PAT mRNAs but become more prominent at 30-75 nucleotides. We also analyzed time courses of PAT decay transcriptome-wide and for ~200 immune response mRNAs. This demonstrated accelerated deadenylation in KO cells on PATs <75 nucleotides and phasing features consistent with greater PABP dissociation in the absence of LARP4. Thus, LARP4 shapes PAT profiles throughout mRNA lifespan and with impact on mRNA decay at short lengths known to sensitize PABP dissociation in response to deadenylation machinery.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD000412-31 PGD)
- Richard J Maraia
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Nahum Sonenberg, McGill University, Canada
Version history
- Received: June 2, 2020
- Accepted: August 2, 2020
- Accepted Manuscript published: August 3, 2020 (version 1)
- Version of Record published: August 7, 2020 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
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Single molecule poly(A) tail-seq shows LARP4 opposes deadenylation through mRNA lifespan with most impact on short tails
eLife 9:e59186.
https://doi.org/10.7554/eLife.59186
Further reading
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Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.
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