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The SMAD2/3 Interactome Reveals That Tgfβ Controls M6a Mrna

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The SMAD2/3 Interactome Reveals That Tgfβ Controls M6a Mrna 6 1 The SMAD2/3 interactome reveals that TGFβ controls m A 2 mRNA methylation in pluripotency 3 4 Alessandro Bertero1,*,†, Stephanie Brown1,*, Pedro Madrigal1,2, Anna Osnato1, Daniel 5 Ortmann1, Loukia Yiangou1, Juned Kadiwala1, Nina C. Hubner3, Igor Ruiz de los Mozos4, 6 Christoph Sadee4, An-Sofie Lenaerts1, Shota Nakanoh1, Rodrigo Grandy1, Edward Farnell5, 7 Jernej Ule4, Hendrik G. Stunnenberg3, Sasha Mendjan1,‡, and Ludovic Vallier1,2,#. 8 9 1 Wellcome Trust - MRC Cambridge Stem Cell Institute Anne McLaren Laboratory and 10 Department of Surgery, University of Cambridge, UK. 11 2 Wellcome Trust Sanger Institute, Hinxton UK. 12 3 Department of Molecular Biology, Radboud University Nijmegen, The Netherlands. 13 4 Francis Crick Institute and Department of Molecular Neuroscience, University College 14 London, UK. 15 5 Department of Pathology, University of Cambridge, UK. 16 17 * These authors contributed equally to this work. 18 † Current address: Department of Pathology, University of Washington, Seattle, WA, USA. 19 ‡ Current address: Institute of Molecular Biotechnology, Vienna, Austria. 20 # Corresponding author ([email protected]). 1 21 The TGFβ pathway plays an essential role in embryonic development, organ 22 homeostasis, tissue repair, and disease1,2. This diversity of tasks is achieved through the 23 intracellular effector SMAD2/3, whose canonical function is to control activity of target 24 genes by interacting with transcriptional regulators3. Nevertheless, a complete 25 description of the factors interacting with SMAD2/3 in any given cell type is still lacking. 26 Here we address this limitation by describing the interactome of SMAD2/3 in human 27 pluripotent stem cells (hPSCs). This analysis reveals that SMAD2/3 is involved in 28 multiple molecular processes in addition to its role in transcription. In particular, we 29 identify a functional interaction with the METTL3-METTL14-WTAP complex, which 30 deposits N6-methyladenosine (m6A)4. We uncover that SMAD2/3 promotes binding of 31 the m6A methyltransferase complex onto a subset of transcripts involved in early cell 32 fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, 33 including the pluripotency factor NANOG, thereby poising them for rapid 34 downregulation upon differentiation to enable timely exit from pluripotency. 35 Collectively, these findings reveal the mechanism by which extracellular signalling can 36 induce rapid cellular responses through regulations of the epitranscriptome. These novel 37 aspects of TGFβ signalling could have far-reaching implications in many other cell types 38 and in diseases such as cancer5. 2 39 Main 40 41 Activin and Nodal, two members of the TGFβ superfamily, play essential roles in cell fate 42 decision in hPSCs6–8. Activin/Nodal signalling is necessary to maintain pluripotency, and its 43 inhibition drives differentiation toward the neuroectoderm lineage6,9,10. Activin/Nodal also 44 cooperates with BMP and WNT to drive mesendoderm specification11–14. Thus, we used hPSC 45 differentiation into definitive endoderm as a model system to interrogate the SMAD2/3 46 interactome during a dynamic cellular process. For that we developed an optimized SMAD2/3 47 co-immunoprecipitation (co-IP) protocol compatible with mass-spectrometry analyses 48 (Extended Data Fig. 1a-b and Supplementary Discussion). This method allowed a 49 comprehensive and unbiased examination of the proteins interacting with SMAD2/3 for the 50 first time in any given cell type. By examining human embryonic stem cells (hESCs) and 51 hESCs induced to differentiate towards endoderm (Fig. 1a), we identified 89 SMAD2/3 52 partners (Fig. 1b, Extended Data Fig. 1c-d, and Supplementary Table 1). Of these, only 11 53 factors were not shared between hESCs and endoderm differentiating cells (Extended Data 54 Fig. 1e), suggesting that the SMAD2/3 interactome is largely conserved across these two 55 lineages (Supplementary Discussion). Importantly, this list included known SMAD2/3 56 transcriptional and epigenetic cofactors (including FOXH1, SMAD4, SNON, SKI, EP300, 57 SETDB1, and CREBBP3), which validated our method. Furthermore, we performed functional 58 experiments on FOXH1, EP300, CREBBP, and SETDB1, which uncovered the essential 59 function of these SMAD2/3 transcriptional and epigenetic cofactors in hPSC fate decisions 60 (Extended Data Fig. 2 and 3, and Supplementary Discussion). 61 62 Interestingly, our proteomic experiments also revealed that SMAD2/3 interacts with 63 complexes involved in functions that have never been associated with TGFβ signalling (Fig. 3 64 1b and Extended Data Fig. 1f), such as ERCC1-XPF (DNA repair) and DAPK3-PAWR 65 (apoptosis). Most notably, we identified several factors involved in mRNA processing, 66 modification, and degradation (Fig. 1b), such as the METTL3-METTL14-WTAP complex 67 (deposition of N6-methyladenosine, or m6A), the PABP-dependent poly(A) nuclease complex 68 hPAN (mRNA decay), the cleavage factor complex CFIm (pre-mRNA 3’ end processing), and 69 the NONO-SFPQ-PSPC1 factors (RNA splicing and nuclear retention of defective RNAs). 70 Overall, these results suggest that SMAD2/3 could be involved in a large number of biological 71 processes in hPSCs, which include not only transcriptional and epigenetic regulations, but also 72 novel “non-canonical” molecular functions. 73 74 To further explore this hypothesis, we investigated the interplays between Activin/Nodal and 75 m6A deposition. m6A is the most common RNA modification, regulating multiple aspects of 76 mRNA biology including decay and translation4,15–19. However, whether this is a dynamic 77 event that can be modulated by extracellular cues remains to be established. Furthermore, 78 while m6A is known to regulate hematopoietic stem cells20,21 and the transition between the 79 naïve and primed pluripotency states22,23, its function in hPSCs and during germ layer 80 specification is unclear. We first validated the interaction of SMAD2/3 with METTL3- 81 METTL14-WTAP using co-IP followed by Western Blot in both hESCs and human induced 82 pluripotent stem cells (hiPSCs; Fig. 2a and Extended Data Fig. 4a-b). Interestingly, inhibition 83 of SMAD2/3 phosphorylation blocked this interaction (Fig. 2b and Extended Data Fig. 4c). 84 Proximity ligation assays (PLA) also demonstrated that the interaction occurs at the nuclear 85 level (Fig. 2c-d). These observations suggest that SMAD2/3 and the m6A methyltransferase 86 complex interact in an Activin/Nodal signalling-dependent fashion. 87 4 88 To investigate the functional relevance of this interaction, we assessed the transcriptome-wide 89 effects of Activin/Nodal inhibition on the deposition of m6A by performing nuclear-enriched 90 m6A methylated RNA immunoprecipitation followed by deep sequencing (NeMeRIP-seq; 91 Extended Data Fig. 5a-d, and Supplementary Discussion). In agreement with previous 92 reports17,19,24, deposition of m6A onto exons was enriched around stop codons and 93 transcription start sites, and occurred at a motif corresponding to the m6A consensus sequence 94 (Extended Data Fig. 5e-g). Assessment of differential m6A deposition revealed that 95 Activin/Nodal inhibition predominantly resulted in reduced m6A levels in selected transcripts 96 (Supplementary Table 2; average absolute log2 fold-change of 0.56 and 0.35 for m6A decrease 97 and increase, respectively). Decrease in m6A deposition was predominantly observed on 98 peaks located near to stop codons (Extended Data Fig. 5h), a location which has been reported 99 to decrease the stability of mRNAs16,24,25. Interestingly, transcripts showing reduced m6A 100 levels after Activin/Nodal inhibition largely and significantly overlapped with genes bound by 101 SMAD2/3 (Extended Data Fig. 5i), including well-known transcriptional targets such as 102 NANOG, NODAL, LEFTY1, and SMAD7 (Fig. 2e and Extended Data Fig. 5j). Accordingly, 103 Activin/Nodal-sensitive m6A deposition was largely associated with transcripts rapidly 104 decreasing during the exit from pluripotency triggered by Activin/Nodal inhibition (Extended 105 Data Fig. 6a). Transcripts behaving in this fashion were enriched in pluripotency regulators 106 and in factors involved in the Activin/Nodal signalling pathway (Supplementary Table 3). On 107 the other hand, the expression of a large number of developmental regulators associated to 108 Activin/Nodal-sensitive m6A deposition remained unchanged following Activin/Nodal 109 inhibition (Extended Data Fig. 6a-c and Supplementary Table 3). Considered together, these 110 findings establish that Activin/Nodal signalling can regulate m6A deposition on a number of 111 specific transcripts. 112 5 113 We then examined the underlying molecular mechanisms. RNA immunoprecipitation 114 experiments on nuclear RNAs showed that inhibition of Activin/Nodal signalling impaired 115 binding of WTAP to multiple m6A-marked transcripts including NANOG and LEFTY1 (Fig. 116 2f and Extended Data Fig. 4d-e), while SMAD2/3 itself interacted with such transcripts in the 117 presence of Activin/Nodal signalling (Fig. 2g and Extended Data Fig. 4e). Thus, SMAD2/3 118 appears to promote the recruitment of the m6A methyltransferase complex onto nuclear 119 RNAs. Interestingly, recent reports have established that m6A deposition occurs co- 120 transcriptionally and involves nascent pre-RNAs16,26,20. Considering the broad overlap 121 between SMAD2/3 transcriptional targets and transcripts showing Activin/Nodal-sensitive 122 m6A deposition (Extended Data Fig 5i),
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