Functional Separation of mRNA Domains Coordinates Pluripotent Cell Behavior

  1. Ze Yang
  2. Shaoyi Ji
  3. Kristina Ivanov
  4. Priyanka Kadav
  5. Mengmeng Song
  6. Leonardi Gozali
  7. Sophie Parsa
  8. Barry Behr
  9. Mary Hynes

  • Curated by eLife

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    eLife Assessment

    This study provides fundamental insights by demonstrating that the Nanog mRNA coding sequence (CDS) and 3′UTR domains are spatially segregated and functionally distinct in pluripotent stem cells and blastocysts, with 3′UTR-enriched border cells primarily influencing morphogenesis and CDS-enriched inner cells largely regulating transcription and epigenetic programs. The work opens a novel conceptual avenue for understanding how separable mRNA domains can differentially control cell behavior and differentiation. However, the evidence is incomplete, as key aspects of the molecular nature, biogenesis, and precise characterization of the separated 3′UTR and CDS RNA species, as well as causal links between their perturbation and the observed phenotypes (e.g., via rescue and deeper characterization of 3′UTR elements), remain to be fully established.

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Abstract

Differential expression of mRNA coding sequences (CDSs) and 3-prime untranslated regions (UTRs) is widespread, yet whether these domains contribute independently to cellular function remains unclear. Here, using Nanog as a model, we find that Nanog mRNA domain usage is spatially organized in mouse and human pluripotent cells and in the blastocyst, with cells enriched in 3-prime UTR transcripts marking colony borders and cells enriched for the Nanog CDS located interior. Functional perturbation reveals a marked asymmetry between mRNA domains. Loss of the Nanog 3-prime UTR leads to defects in colony architecture, cell spreading and morphogenetic behavior, accompanied by decreased extracellular matrix modeling gene expression and ROCK dependent cytoskeletal organization. In contrast, deletion of the Nanog CDS primarily disrupts epithelial polarity-associated transcriptional programs and the expression of chromatin regulators, consistent with a dominant role for the Nanog protein in transcriptional and epigenetic control. These domain specific effects are not redundant but instead reflect separable regulatory activities encoded within a single transcript. Together, these findings demonstrate that distinct regions of a single mRNA can encode separable and asymmetric biological functions, revealing mRNA domain usage as a distinct regulatory layer through which genes can encode multiple biological outputs beyond protein coding capacity.

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  1. eLife

    eLife Assessment

    This study provides fundamental insights by demonstrating that the Nanog mRNA coding sequence (CDS) and 3′UTR domains are spatially segregated and functionally distinct in pluripotent stem cells and blastocysts, with 3′UTR-enriched border cells primarily influencing morphogenesis and CDS-enriched inner cells largely regulating transcription and epigenetic programs. The work opens a novel conceptual avenue for understanding how separable mRNA domains can differentially control cell behavior and differentiation. However, the evidence is incomplete, as key aspects of the molecular nature, biogenesis, and precise characterization of the separated 3′UTR and CDS RNA species, as well as causal links between their perturbation and the observed phenotypes (e.g., via rescue and deeper characterization of 3′UTR elements), remain to be fully established.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    There is evidence that some genes encode mRNAs from which separate processed transcripts may arise, separating the coding sequence (CDS) from the 3'-UTR, and with both mRNA elements remaining stable in the cell. However, the functional consequences of these mRNA fragments have not been firmly established. In the manuscript by Yang et al., the authors probe the mRNA domain architecture of Nanog in the context of embryonic stem cell colonies and blastocysts. The authors detect spatial separation of Nanog CDS-containing mRNA from abundant Nanog 3'-UTR RNAs depending on the cell position in 2D embryonic stem cell colonies or in blastocysts.

    Strengths:

    The phenotypic analyses of the Nanog mRNA hold promise for revealing distinct roles for the Nanog encoded protein and a separate RNA encompassing the Nanog 3'-UTR.

    Weaknesses:

    There are a number of questions about the molecular nature of the mRNA species that the authors should address in order for the results to be firmly established, as noted below.

    (1) It is not clear how the authors verified that their probes are specific for Nanog CDS or 3'-UTR regions. Especially for the 3'-UTR probe, it is confusing why colonies show green only regions, suggesting only the CDS is present. I would expect the CDS and 3'-UTR probes to colocalize in the interior cells. Is it possible that the 3'-UTR probe is targeting another RNA?

    (2) It would help for the authors to include a graphic similar to Figure 3, Figure Supplement 1A, that diagrams the location of the CDS and 3'-UTR probes (this should also be done for Oct4 and Sox2). This graphic could also show all potential polyadenylation signals.

    (3) I think, based on the fluorescence patterns, there is evidence that the signal for the Nanog 3'-UTR probe is nuclear (images with DAPI staining), but this is not commented on that I could find. This should be discussed, as nuclear retention has implications for the noncoding function of the 3'-UTR fragment.

    (4) Figure 2, Figure Supplement 1A needs a better explanation. It's not clear how the reads map to the different regions of the Nanog mature mRNA. The authors should show examples at different ratios of CDS to 3'-UTR. Do the reads have a sharp boundary at the junction of where the isolated 3'-UTR is thought to occur?

    (5) I looked in the Zenbu browser at human NANOG CAGE mapping in the FANTOM5 dataset. I could not see evidence for substantial capping of a 3'-UTR fragment when filtering for embryonic cell types. Given the strong signal for the 3'-UTR in border cells, I would expect to see evidence for capping if the RNA were indeed capped. This suggests that if it exists, it is likely uncapped and (as noted in point 3) is likely nuclear retained.

    (6) Are there predicted polyadenylation signals near the end of the CDS that would generate a short 3'-UTR, and are these signals conserved across mammals?

    (7) It would help to see a zoomed-in view of the region targeted by one of the guide RNAs in the 3'-UTR, and where that site is relative to the polyadenylation signal. Is the polyadenylation signal upstream, i.e., CDS proximal?

    (8) A final note, the use of green and red together will be challenging for those who are colorblind. Providing a different false color palette would be helpful.

    I am refraining from comments on the cell biology and morphological insights, as they are remote from my core expertise.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    This manuscript shows that the coding sequence (CDS) and 3' untranslated region (3'UTR) of mRNA transcripts from the Nanog gene have distinct expression patterns and functions. In both human and mouse embryonic stem cells colonies and blastocysts, these domains are spatially segregated, with 3'UTR-enriched cells occupying the borders and CDS-enriched cells residing in the interior. CDS mRNA expression is correlated with the expected regulation of transcription and epigenetics associated with the Nanog protein. Interestingly, expression of the 3'UTR appears to play an independent role in cell behavior and colony morphogenesis. Indeed, deletion of the 3'UTR causes specific defects in cell spreading and protrusive activity, with alteration in the localization of adhesion and cytoskeleton-associated proteins. Remarkably, a large proportion of those defects are rescued upon ROCK inhibition. Deletion of either Nanog CDS or 3'UTR leads to distinct modifications in the differentiation competence.

    Strengths:

    The independent role of 3'UTR mRNA domains, although identified in neurosciences a couple of years ago, is a novel and exciting field relatively unexplored in early development.

    The manuscript offers a multilayer series of experiments, in ES cells colony, blastocysts, and embryoid bodies, including imaging, -omics, genetic and pharmacological challenges, and differentiation experiments, thereby unveiling very convincingly the role of Nanog 3'UTR in morphogenesis.

    Weaknesses:

    The pathways leading to the generation of those distinct transcript domains are unknown. Although the functional differential roles are well demonstrated, whether the expression patterns are a cause or a consequence of the cells' localisation in the embryo remains to be explored.

  4. eLife

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, Yang et al reported distinct functions of the protein-coding sequence (CDS) and the 3' untranslated region (UTR) in the Nanog mRNA in pluripotent stem cells. They first observed different localization patterns for the CDS and 3' UTR in embryonic stem cells and in blastocyst embryos, and this pattern correlates with cell populations in different pluripotent states based on single-cell sequencing data. To characterize the potentially distinct functions of these regions, the authors generated knockout (KO) cell lines in which either the CDS or the 3' UTR was genetically ablated. These deletions led to different phenotypes in multiple assays. These results provided evidence that the CDS and 3' UTR of an mRNA could have distinct functions. Although these results are potentially interesting, several questions need to be addressed before the validity of their conclusion can be confirmed.

    Strengths:

    This study provides evidence for distinct functions of the protein-coding sequence and 3' untranslated region of an mRNA in pluripotent stem cells. The concept could be more broadly applied.

    Weaknesses:

    The initial observation (distinct localization of CDS and 3' UTRs) and the causal relationship between the KO and phenotype need further validation.

    Major points:

    (1) The authors showed distinct localization patterns of the CDS and 3' UTRs in human and mouse ESCs and blastocysts, and the overlap between their signals was minimal (Figure 1). Does this mean that the CDS and 3' UTR RNAs exist separately? For example, in cells that only showed signals for 3' UTRs, do these RNAs only contain 3' UTRs and lack CDS? Was this confirmed by RNA-seq experiments? If so, how are they generated (i.e., by transcription from a novel promoter or partial degradation of the full-length mRNAs)? This is a key question. Without a clear characterization of these RNAs, the rest of the study cannot be substantiated.

    (2) To confirm that the phenotypes of CDS or 3' UTR KO cells were caused by the deleted regions instead of other artifacts, rescue experiments should be performed.

    (3) As over-expression of the 3' UTR showed a phenotype, important regions within it should be identified, and also the possibility that the 3' UTR contains open reading frame(s) and is translated should be tested.

  5. eLife

    Author response:

    eLife Assessment

    This study provides fundamental insights by demonstrating that the Nanog mRNA coding sequence (CDS) and 3′UTR domains are spatially segregated and functionally distinct in pluripotent stem cells and blastocysts, with 3′UTR-enriched border cells primarily influencing morphogenesis and CDS-enriched inner cells largely regulating transcription and epigenetic programs. The work opens a novel conceptual avenue for understanding how separable mRNA domains can differentially control cell behavior and differentiation. However, the evidence is incomplete, as key aspects of the molecular nature, biogenesis, and precise characterization of the separated 3′UTR and CDS RNA species, as well as causal links between their perturbation and the observed phenotypes (e.g., via rescue and deeper characterization of 3′UTR elements), remain to be fully established.

    We thank the editors and the three reviewers for their careful and constructive engagement with our manuscript. We greatly appreciate the reviewers’ recognition of the conceptual significance of the study and their thoughtful suggestions for strengthening the mechanistic and molecular characterization of the work. We have carefully considered all points raised and outline below the revisions planned for the revised manuscript.

    The phenomenon of differential CDS and 3’UTR expression is not unique to Nanog. Independent 3’UTR and CDS expression and differential CDS/3’UTR usage has been observed across multiple genes, tissues, and developmental contexts, including genome-wide (Mercer et al., 2011) and transcriptome scale studies (Kocabas et al., 2025, Ji et al., 2021). Prior studies have proposed that isolated 3’UTRs may arise through regulated RNA processing pathways coupled to exonucleolytic degradation and, in some cases, recapping mechanisms (Malka et al, 2017, Haberman et al., 2024). While the precise molecular mechanisms underlying isolated Nanog CDS and 3’UTR generation remain unresolved, our observations (contained here) support regulated RNA processing models. Our original submission included a brief discussion of this topic; however the revised manuscript will include substantially expanded analyses and discussion of the generation of isolated Nanog CDS and 3’UTR species.

    The revised manuscript will address the major concerns regarding:

    (1) The molecular nature, biogenesis, and precise characterization of the separated 3′UTR and CDS mRNA species

    (2) The causal relationship between perturbation of these RNA species and the observed phenotypes, including additional rescue experiments and deeper computational characterization of putative, functional 3′UTR elements.

    Specifically:

    (A) New supplementary analyses and schematics designed to further clarify the conceptual and mechanistic framework of the study, including:

    (i) Computational examination of the Nanog 3’UTR across all reading frames for open reading frames (ORFs).

    (ii) As suggested by Reviewers 1 and 3, single cell traces of Nanog mRNA expression from the full-length mESC dataset used in this study, illustrating distinct transcript isoforms and CDS/3’UTR expression patterns across individual cells, complementing the color-coded tSNE analyses currently presented in Fig. 2.

    (iii) Expanded schematic model and analyses addressing possible mechanisms underlying the generation of isolated Nanog CDS and 3’UTR enriched RNA species, including transcript architecture, predicted RNA structural barriers, and exonucleolytic processing models.

    (iv) Expanded discussion of the predominantly nuclear localization of the Nanog 3’UTR signal and its implications for transcript biogenesis, processing, and potential noncoding functions.

    (B) Correction of all minor labeling errors.

    (C) Additional experimental analyses, including:

    - Expansion of Nanog 3’UTR overexpression and rescue experiments to include cell spreading assays.

    - Expanded analysis of the effects of ROCK pathway inhibitors on colony morphology and cytoskeletal organization.

    - Examination of the ability of ROCK inhibition to restore normal embryoid body formation.

    Collectively, these planned revisions are intended to strengthen the mechanistic framing, molecular characterization, and broader significance of the study while clarifying the interpretation and scope of the conclusions.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    There is evidence that some genes encode mRNAs from which separate processed transcripts may arise, separating the coding sequence (CDS) from the 3'-UTR, and with both mRNA elements remaining stable in the cell. However, the functional consequences of these mRNA fragments have not been firmly established. In the manuscript by Yang et al., the authors probe the mRNA domain architecture of Nanog in the context of embryonic stem cell colonies and blastocysts. The authors detect spatial separation of Nanog CDS-containing mRNA from abundant Nanog 3'-UTR RNAs depending on the cell position in 2D embryonic stem cell colonies or in blastocysts.

    Strengths:

    The phenotypic analyses of the Nanog mRNA hold promise for revealing distinct roles for the Nanog encoded protein and a separate RNA encompassing the Nanog 3'-UTR.

    Weaknesses:

    There are a number of questions about the molecular nature of the mRNA species that the authors should address in order for the results to be firmly established, as noted below.

    (1) It is not clear how the authors verified that their probes are specific for Nanog CDS or 3'-UTR regions. Especially for the 3'-UTR probe, it is confusing why colonies show green only regions, suggesting only the CDS is present. I would expect the CDS and 3'-UTR probes to colocalize in the interior cells. Is it possible that the 3'-UTR probe is targeting another RNA?

    We thank the reviewer for raising the important question of probe specificity. We realize that the data that underlying this concern is the absence of colocalizing between CDS and 3’UTR probes in colony border cells.

    The absence of CDS/3’UTR colocalization in colony border cells is not due to probe failure but instead reflects the principal observation underlying the study. If Nanog CDS and 3’UTR sequences were present exclusively as intact full-length transcripts in a strict stoichiometric ratio, Nanog positive cells would be expected to be positive for both probes (appearing yellow). Instead, border cells exhibit strong 3’UTR signal with minimal or absent CDS signal, while adjacent interior cells show the opposite pattern.

    The fact that both probes robustly detect signal within the same sample but in spatially distinct cell populations, argues that both probes are functional and that the observed differential localization reflects genuine biological differences in levels of transcript components.

    The CDS probe targets ~300 bp within the coding region, while the 3’UTR probe targets ~300 bp within the proximal region of the Nanog 3’UTR. Hybridization specificity was validated as described in the Methods and in our previous studies (Kocabas et al 2015; Ji et al 2021), including negative controls. We additionally now provide a supplemental figure (New Figure 1-figure supplement 2A), highlighting that the Nanog 3’UTR and CDS probes label cell populations distinct from each other, further indicating their specificity.

    In addition, full-length scRNA seq datasets from both mouse and human ESCs demonstrate differential CDS/3’UTR expression patterns for Nanog and many other genes. To further clarify this point, the revised manuscript will include single cell transcript traces from mESCs illustrating the distinct Nanog isoforms detected across individual cells (New Figure 2-figure supplement 1A)

    (2) It would help for the authors to include a graphic similar to Figure 3, Figure Supplement 1A, that diagrams the location of the CDS and 3'-UTR probes (this should also be done for Oct4 and Sox2). This graphic could also show all potential polyadenylation signals.

    We agree that additional schematic clarification would improve readability. The revised manuscript will include schematics showing the locations of the CDS and 3’UTR probes for Nanog, Sox2 and Oct4 (New Fig. 1- figure supplement 1A).

    (3) I think, based on the fluorescence patterns, there is evidence that the signal for the Nanog 3'-UTR probe is nuclear (images with DAPI staining), but this is not commented on that I could find. This should be discussed, as nuclear retention has implications for the noncoding function of the 3'-UTR fragment.

    The reviewer is correct that the Nanog 3’UTR signal mostly nuclear. Whie this was noted in (the original) Figure 1-figure supplement 2A, we agree that it is possible that mechanistic and functional implications were not sufficiently discussed in the original manuscript. The revised manuscript will include expanded discussion of the relationship between nuclear localization transcript processing, and potential noncoding functions of isolated Nanog 3’UTR species

    (4) Figure 2, Figure Supplement 1A needs a better explanation. It's not clear how the reads map to the different regions of the Nanog mature mRNA. The authors should show examples at different ratios of CDS to 3'-UTR. Do the reads have a sharp boundary at the junction of where the isolated 3'-UTR is thought to occur?

    We thank the reviewer for this suggestion. The revised manuscript will include new single cell read maps across the Nanog locus from full length mESC scRNA-seq datasets (New Figure 2-figure supplement 1A), illustrating distinct CDS enriched and 3’UTR enriched transcript isoforms across individual cells.

    These analyses indicate that some CDS dominant transcripts contain 3’UTR sequence, while many appear to contain little or no detectable 3’UTR sequence. Conversely, many 3’UTR enriched transcripts contain only minimal or truncated CDS sequence. Importantly full CDS and 3’UTR mRNA components are frequently not present in a strict 1:1 ratio, either within individual cells, or across cell populations.

    The revised manuscript will also include expanded supplementary analyses integrating transcript architecture, predicted RNA structural barriers, polyadenylation analysis, and single cell coverage patterns to further examine possible mechanisms underlying the generation of isolated Nanog CDS and 3’UTR species (New Figure 2-figure supplement 1B,C).

    (5) I looked in the Zenbu browser at human NANOG CAGE mapping in the FANTOM5 dataset. I could not see evidence for substantial capping of a 3'-UTR fragment when filtering for embryonic cell types. Given the strong signal for the 3'-UTR in border cells, I would expect to see evidence for capping if the RNA were indeed capped. This suggests that if it exists, it is likely uncapped and (as noted in point 3) is likely nuclear retained.

    Prior studies have reported isolated uncapped and recapped 3’UTR species in multiple systems (Malka et al, 2017; Haberman et al, 2024). We agree that the predominantly nuclear localization and lack of a strong CAGE signal for Nanog are important observations and will expand discussion of these points in the revised manuscript.

    (6) Are there predicted polyadenylation signals near the end of the CDS that would generate a short 3'-UTR, and are these signals conserved across mammals?

    Computational analysis of the mouse Nanog 3'UTR identifies a single canonical PAS (AATAAA) at position 1074, located at the 3’ end of the annotated 3’UTR and this terminal PAS is conserved across mammals. These analyses will be included as a supplementary figure and discussed further in the revised manuscript section addressing Nanog transcript biogenesis.

    (7) It would help to see a zoomed-in view of the region targeted by one of the guide RNAs in the 3'-UTR, and where that site is relative to the polyadenylation signal. Is the polyadenylation signal upstream, i.e., CDS proximal?

    This will be provided in the revised manuscript (New Figure 2-figure supplement 1C,i) Two guide RNAs were used to generate the Nanog 3’UTR deletions. The downstream guide is upstream of the terminal polyadenylation signal at nt 1074 to preserve polyadenylation of the remaining Nanog CDS containing transcript.

    Consistent with this, all Nanog 3’UTR knockout lines retain normal Nanog protein levels. The revised manuscript will include supplementary schematics showing guide RNA positions relative to the CDS, 3’UTR probes, and terminal PAS.

    (8) A final note, the use of green and red together will be challenging for those who are colorblind. Providing a different false color palette would be helpful.

    We appreciate this attention to accessibly. The red/green color combination was chosen to provide the highest contrast between CDS and 3’UTR signals in the in situ hybridization experiments, which is important for visualizing their differential spatial localization. We will ensure that figure legends clearly indicate channel assignments throughout the manuscript.

    I am refraining from comments on the cell biology and morphological insights, as they are remote from my core expertise.

    Reviewer #2 (Public review):

    Summary:

    This manuscript shows that the coding sequence (CDS) and 3' untranslated region (3'UTR) of mRNA transcripts from the Nanog gene have distinct expression patterns and functions. In both human and mouse embryonic stem cells colonies and blastocysts, these domains are spatially segregated, with 3'UTR-enriched cells occupying the borders and CDS-enriched cells residing in the interior. CDS mRNA expression is correlated with the expected regulation of transcription and epigenetics associated with the Nanog protein. Interestingly, expression of the 3'UTR appears to play an independent role in cell behavior and colony morphogenesis. Indeed, deletion of the 3'UTR causes specific defects in cell spreading and protrusive activity, with alteration in the localization of adhesion and cytoskeleton-associated proteins. Remarkably, a large proportion of those defects are rescued upon ROCK inhibition. Deletion of either Nanog CDS or 3'UTR leads to distinct modifications in the differentiation competence.

    Strengths:

    The independent role of 3'UTR mRNA domains, although identified in neurosciences a couple of years ago, is a novel and exciting field relatively unexplored in early development.

    The manuscript offers a multilayer series of experiments, in ES cells colony, blastocysts, and embryoid bodies, including imaging, -omics, genetic and pharmacological challenges, and differentiation experiments, thereby unveiling very convincingly the role of Nanog 3'UTR in morphogenesis.

    Weaknesses:

    The pathways leading to the generation of those distinct transcript domains are unknown. Although the functional differential roles are well demonstrated whether the expression patterns are a cause or a consequence of the cells' localization in the embryo remains to be explored.

    We thank the reviewer for these thoughtful comments and for recognizing the potential significance of independent 3’UTR functions in early developmental systems.

    Regarding the mechanisms underlying generation of distinct CDS and 3’UTR transcript domains, the revised manuscript will include new supplementary analyses and schematic models addressing possible Nanog transcript processing pathways, as outlined above.

    We agree that the relation between spatial location and Nanog 3’UTR expression is an important question. Specifically, it remains unclear whether cells first acquire high Nanog 3’UTR expression and subsequently localize to the colony border or whether border position itself promotes high Nanog 3’UTR expression.

    Our current data suggest that both processes may contribute. Deletion of the Nanog 3’UTR does not prevent colonies from establishing border/interior pattern, indicating that high Nanog 3’UTR is not strictly required for border pattern itself. At the same time, Nanog 3’UTR overexpression and rescue experiments increased the likelihood of border localization, suggesting that elevated Nanog 3’UTR expression promotes behaviors associated with border occupancy.

    Reviewer #3 (Public review):

    Summary:

    In this manuscript, Yang et al reported distinct functions of the protein-coding sequence (CDS) and the 3' untranslated region (UTR) in the Nanog mRNA in pluripotent stem cells. They first observed different localization patterns for the CDS and 3' UTR in embryonic stem cells and in blastocyst embryos, and this pattern correlates with cell populations in different pluripotent states based on single-cell sequencing data. To characterize the potentially distinct functions of these regions, the authors generated knockout (KO) cell lines in which either the CDS or the 3' UTR was genetically ablated. These deletions led to different phenotypes in multiple assays. These results provided evidence that the CDS and 3' UTR of an mRNA could have distinct functions. Although these results are potentially interesting, several questions need to be addressed before the validity of their conclusion can be confirmed.

    Strengths:

    This study provides evidence for distinct functions of the protein-coding sequence and 3' untranslated region of an mRNA in pluripotent stem cells. The concept could be more broadly applied.

    Weaknesses:

    The initial observation (distinct localization of CDS and 3' UTRs) and the causal relationship between the KO and phenotype need further validation.

    Major points:

    (1) The authors showed distinct localization patterns of the CDS and 3' UTRs in human and mouse ESCs and blastocysts, and the overlap between their signals was minimal (Figure 1). Does this mean that the CDS and 3' UTR RNAs exist separately? For example, in cells that only showed signals for 3' UTRs, do these RNAs only contain 3' UTRs and lack CDS? Was this confirmed by RNA-seq experiments? If so, how are they generated (i.e., by transcription from a novel promoter or partial degradation of the full-length mRNAs)? This is a key question. Without a clear characterization of these RNAs, the rest of the study cannot be substantiated.

    We thank the reviewer for raising this important question, which overlaps substantially with several key points raised by Reviewer #1 concerning the molecular nature and characterization of the Nanog CDS and 3’UTR species.

    Colony border cells exhibit strong Nanog 3’UTR signal with minimal detectable CDS signal, while adjacent interior cells show the reciprocal pattern. These observations strongly suggest the existence of distinct Nanog transcript species rather than exclusively full-length transcripts containing stoichiometric amounts of both CDS and 3’UTR sequence.

    This conclusion is independently supported by full-length Smart-seq2 scRNA seq datasets from both mouse and human ESCs, which provide transcript coverage across both CDS and 3’UTR regions.

    (2) To confirm that the phenotypes of CDS or 3' UTR KO cells were caused by the deleted regions instead of other artifacts, rescue experiments should be performed.

    Rescue experiments were included in the original submission (Fig. 4). The revised manuscript will expand these analyses to include cell spreading. We will also include additional ROCK pathway modulation experiments.

    (3) As over-expression of the 3' UTR showed a phenotype, important regions within it should be identified, and also the possibility that the 3' UTR contains open reading frame(s) and is translated should be tested.

    The revised manuscript will also include supplementary computational analyses of the Nanog 3’UTR, including open reading frame prediction, Kozak scoring, and evolutionary conservation analysis. (New Figure 2-figure supplement 1B). These analyses identify no evidence for strongly supported coding potential within the 3’UTR. Further, isolated Nanog 3’UTR transcripts are largely confined to the nucleus, making active translation unlikely.

    The revised manuscript will include new supplementary analyses addressing Nanog transcript structure and possible biogenesis mechanisms (New Figure 2-figure supplement 1C).

    References:

    ViennaRNA/RNA fold – Lorenz et al 2011 Algorithms Mol Biol 6:26- RNA Secondary Structure stem loop, minimum free energy (MFE) prediction

    NCBI BLASTP- Altschul et al (1990) J Mol Biol 215:403- ORF conservation, protein sequence similarity search

    NCBI Entrez/Biohthon- Cock et al (2009) Bioinformatics 25:1422- sequence retrieval

    PhastCons/UCSC multiz alignments- Siepel et al (2005) Genome Res 15:1034- evolutionary conservation scoring

    UCSC Genome Browser- Kent et al. (2002) Genome Res 12:996-1006- conservation track access

    Eaton et al (2020) Mol Cell 78:439- Stall model

    Brannan et al (2012) Genes Dev 26:2621-Stall model

    Addition to Methods.

    ORFs (≥10 amino acids) were identified in all three forward frames according to Kozak (1987). Evolutionary conservation was assessed by BLASTP (Altschul et al., 1990) against RefSeq proteins. Poly(A) signals were identified by pattern matching for canonical and non-canonical hexamers. Conserved sequence blocks were obtained from UCSC PhastCons tracks (Siepel et al., 2005). RNA secondary structures were predicted using ViennaRNA RNAfold (Lorenz et al., 2011) with a sliding 80-nt window. The stall model for isolated transcript generation follows Eaton et al. (2020).

  6. Version published to 10.1101/2022.03.15.484504 on bioRxiv