Poised PABP–RNA hubs implement signal-dependent mRNA decay in development

Signaling pathways drive cell fate transitions largely by changing gene expression. However, the mechanisms for rapid and selective transcriptome rewiring in response to signaling cues remain elusive. Here we use deep learning to deconvolve both the sequence determinants and the trans-acting regulators that trigger extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase kinase (MEK)-induced decay of the naive pluripotency mRNAs. Timing of decay is coupled to embryo implantation through ERK–MEK phosphorylation of LIN28A, which repositions pLIN28A to the highly A+U-rich 3′ untranslated region (3′UTR) termini of naive pluripotency mRNAs. Interestingly, these A+U-rich 3′UTR termini serve as poly(A)-binding protein (PABP)-binding hubs, poised for signal-induced convergence with LIN28A. The multivalency of AUU motifs determines the efficacy of pLIN28A–PABP convergence, which enhances PABP 3′UTR binding, decreases the protection of poly(A) tails and activates mRNA decay to enable progression toward primed pluripotency. Thus, the signal-induced convergence of LIN28A with PABP–RNA hubs drives the rapid selection of naive mRNAs for decay, enabling the transcriptome remodeling that ensures swift developmental progression.


Statistics
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Software and code
Policy information about availability of computer code Data collection Steady state super-resolution imaging of live ESCs and immunofluorescence images were acquired using a immunoOlympus IX83 microscope equipped with a VT-iSIM super resolution imaging system using MicroManager system.FACS data was acquired using a LSR Fortessa (BD Biosciences, BD FACSDiva Version 9.2.) flow cytometer.RT-qPCR data was acquired using QuantStudio7 (Thermo) and corresponding software.Western blot data was acquired using Amersham Imager 680 blot and gel imager.NGS libraries were sequenced as single end 100bp reads on Illumina HiSeq 4000.iCLIP experiments targeting LIN28A, PABPC1, and PABPC4 were sequenced on NovaSeq platform.Direct RNA sequencing for assessment of polyA-tail length was performed with MinION.

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Sample size
No statistical methods were used to predetermine the sample size.For RNA-seq on cell lines new to this study was determined by prior literature (for instance PMID: 34380047 and PMID: 28945705) and thereby we used similar experimental approaches rather than by power analysis.
Data exclusions One iCLIP sample (LIN28A-S200A_ESC_LIF-CHIR-FGF0220626_MM_2) was excluded from subsequent analyses due to low read coverage.

Replication
The number of replicates used in each experiment are described in the figure legends and/or in the Methods section, as are the statistical tests used including the adjustment methods and alpha values for adjusted P values.All replicates successfully reproduced the presented findings, giving consistent results.LIN28A rescue was replicated in multiple independent cell lines, including multiple LIN28A KO clones were used.Statistical tests are selected appropriately to the analysed data, considering normality, variance, independence (paired or independent tests), and direction of effect (two-sided or one-sided tests).To compare two independent samples, with approximately normal distribution and approximately equal variance, we used two-sided two sample t-test; When the data is approximately normally distributed, but the equal variance criteria is not met, we used two-sided Welch's t-test; When the data did not meet the criteria for normality and/or equal variance, we used the two-sided Mann-Whitney-Wilcoxon rank-sum test.We occasionally employed the non-parametric two-sided Mann-Whitney-Wilcoxon rank-sum test in place of two sample t-test and Welch's t-test, due to fewer underying assumptions.In the figures, we indicate the comparisons of interest and report the exact P values when they are in range between 10e-4 to 10e-10; for P values lower than 10e-10, we label the P values as <10e-10.
Randomization Randomization is not relevant to this study as no randomization is required due to the homogeneous nature of the cell lines.Furthermore, our RNA-seq discovery assays are high-throughput and were initially done in a hypotheses-free analysis.Observations in the RNA-seq were validated and confirmed by biochemical assays to bolster initial observations from the high-throughput assays.

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Immunoflourescence validation experiments were analysed blinded to genotype status.
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used for FACS experiments SSEA1 (MC480, Thermo) SSEA4 (MC813-70, Thermo) Antibodies used for Western blotting LIN28A (A177 Cell Signaling and AF3757, R&D Systems) H3 (Abcam, ab1791) nature portfolio | reporting summary April 2023 For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.
Data Policy information about availability of dataAll manuscripts must include a data availability statement.This statement should provide the following information, where applicable:-Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Sequencing data related to iCLIP, Quantseq and Nanopore direct RNA sequencing experiments for Flag-tagged LIN28A-WT in 2iLIF and FGF2 treated cells, Flagtagged LIN28A-S200A in FGF2 treated cells as well as for iCLIPs of PABPC1 and PABPC4 (in LIN28A KO cells with and without LIN28A overexpression) are available from ENA, with the accession code PRJEB60519.SlamSeq sequencing data, Quantseq data of LIN28A-GFP overexpression in LIN28A KO cells and LIN28A-GFP iCLIP experiments can be retrieved from GEO accession GSE169555.In addition, full data produced by iCLIP analysis pipeline for LIN28A-WT (in 2iL and FGF2 treated cells), LIN28A-S200A (in FGF2 treated cells) as well as for PABPC1 and PABPC4 (in LIN28A KO cells with and without LIN28A overexpression), can be accessed on the iMaps and Flow web-servers: LIN28A iCLIP experiments: https://imaps.goodwright.com/collections/882635250203/;https://app.flow.bio/projects/882635250203/PABPCiCLIP experiments: https://imaps.goodwright.com/collections/340215254997/;https://app.flow.bio/projects/340215254997/Tofacilitate reproduction of our work, we archived key data from iCLIP analysis, used in downstream bioinformatic analyses-crosslink sites, peaks, and motif enrichments from PEKA-on Zenodo (https://zenodo.org/doi/10.5281/zenodo.10054231).Research involving human participants, their data, or biological material Policy information about studies with human participants or human data.See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.