Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids
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eLife assessment
The study integrates proteomic and transcriptomic analyses of human stem cell-derived cortical brain organoids, uncovering posttranscriptional regulatory mechanisms for a specific gene module enriched in ribosomal genes. The data analysis is robust and the evidence supporting the conclusions is compelling. The work provides a valuable resource to developmental neurobiologists and highlights a new level of regulation that may be important in cortical development.
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Abstract
During development of the human cerebral cortex, multipotent neural progenitors generate excitatory neurons and glial cells. Investigations of the transcriptome and epigenome have revealed important gene regulatory networks underlying this crucial developmental event. However, the posttranscriptional control of gene expression and protein abundance during human corticogenesis remains poorly understood. We addressed this issue by using human telencephalic brain organoids grown using a dual reporter cell line to isolate neural progenitors and neurons and performed cell class and developmental stage-specific transcriptome and proteome analysis. Integrating the two datasets revealed modules of gene expression during human corticogenesis. Investigation of one such module uncovered mTOR-mediated regulation of translation of the 5’TOP element-enriched translation machinery in early progenitor cells. We show that in early progenitors partial inhibition of the translation of ribosomal genes prevents precocious translation of differentiation markers. Overall, our multiomics approach proposes novel posttranscriptional regulatory mechanisms crucial for the fidelity of cortical development.
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eLife assessment
The study integrates proteomic and transcriptomic analyses of human stem cell-derived cortical brain organoids, uncovering posttranscriptional regulatory mechanisms for a specific gene module enriched in ribosomal genes. The data analysis is robust and the evidence supporting the conclusions is compelling. The work provides a valuable resource to developmental neurobiologists and highlights a new level of regulation that may be important in cortical development.
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Reviewer #1 (Public Review):
The authors sought to assess how not only RNA but also protein changes across the developmental time course of cortical organoid development. The methods used included reporter lines to label progenitor and neuronal populations, RNA-sequencing, protein quantification using mass spectrometry, and analysis of these results. The primary findings included the identification of RNA sequences that impact translation, the most significant of which was a 5'-TOP cassette that is mediated by mTOR.
Strengths of the paper include strong experimental design, replicates, and images to show the quality of the organoids used in the studies. Additionally, the analysis of elements regulating translation was strong, and the polysome experiments exploring an impact when TSC is deleted were interesting.
Potential limitations …
Reviewer #1 (Public Review):
The authors sought to assess how not only RNA but also protein changes across the developmental time course of cortical organoid development. The methods used included reporter lines to label progenitor and neuronal populations, RNA-sequencing, protein quantification using mass spectrometry, and analysis of these results. The primary findings included the identification of RNA sequences that impact translation, the most significant of which was a 5'-TOP cassette that is mediated by mTOR.
Strengths of the paper include strong experimental design, replicates, and images to show the quality of the organoids used in the studies. Additionally, the analysis of elements regulating translation was strong, and the polysome experiments exploring an impact when TSC is deleted were interesting.
Potential limitations include technical challenges related to the specificity of the reporters, ambiguity about the impact of normalization on the actual protein/RNA data, and potential over-interpretation of the TSC result to encompass all of the mTOR signalings.
The paper validates already observed and documented results in translational regulation whereby RNA does not fully predict protein levels. The impact of the specific examples upon functional significance in cortical development is currently unclear but this work could set the stage for additional future impactful work.
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Reviewer #2 (Public Review):
This work by Sidhaye, Trepte et al. systematically investigates the relationship between transcript and protein abundance across the genome in human neurogenesis. Through analysis of the transcriptome and proteome in brain organoids, they find that for specific gene modules, transcript and protein abundance are highly disconnected. While there are already several anecdotic examples of this phenomenon in the literature, highlighting the role of post-transcriptional gene regulation in corticogenesis, Sidhaye, Trepte et al. for the first time systematically explore the pervasiveness of this phenomenon in a genome-wide manner at different stages of human neurogenesis using a dual reporter cell line to isolate neural progenitor cells and neurons.
The authors then focus on one of the modules that is characterized …
Reviewer #2 (Public Review):
This work by Sidhaye, Trepte et al. systematically investigates the relationship between transcript and protein abundance across the genome in human neurogenesis. Through analysis of the transcriptome and proteome in brain organoids, they find that for specific gene modules, transcript and protein abundance are highly disconnected. While there are already several anecdotic examples of this phenomenon in the literature, highlighting the role of post-transcriptional gene regulation in corticogenesis, Sidhaye, Trepte et al. for the first time systematically explore the pervasiveness of this phenomenon in a genome-wide manner at different stages of human neurogenesis using a dual reporter cell line to isolate neural progenitor cells and neurons.
The authors then focus on one of the modules that is characterized by the enrichment of the 5'TOP (terminal oligopyrimidine) motif in the 5'UTR of transcripts and enriched in ribosomal proteins and translation initiation factors. The authors show that partial inhibition of the translation of ribosomal genes in neural progenitor cells inhibits the translation of differentiation genes, a process that involves mTOR-mediated regulation.
Strength:
The integration of transcriptome and proteome data enables an unbiased systemic analysis revealing gene modules that follow similar trajectories, and as such may share common regulatory principles. For one of the modules, the authors dissect the posttranscriptional regulatory cascade using an elegant combination of fluorescent reporter human pluripotent stem cell lines in combination with gene knockouts.
Overall, the data presented in this work is of a very high standard and supports the conclusions put forward by the authors. The processed omics data sets are made available via a Shiny app web interface for easy access and therefore promote exploration by the scientific community.
Limitations:
This study uses a large range of specific reporter and knockout hPSC lines generated in the context of this work, however, very limited information is provided on these lines. For example, do the lines remain karyotypically normal throughout the targeting procedure? Does reporter gene expression faithfully recapitulate the activity of the promoters controlling their expression? Specifically, it appears that a significant GFP signal is detected within the neuronal layer (Figure 1B) and that there is a much larger double reporter-positive population than expected (Figure S2A).
The authors propose that stress-associated translational regulation takes place in early neural progenitors, involving the sequestration of transcripts in stress granule-like structures. However, given that at least some human brain organoid protocols have been reported to lead to ectopic activation of cellular stress pathways (Bhaduri et al., Nature 2019), it would be desirable to see this aspect of the study confirmed in primary tissue (mouse or human).
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Reviewer #3 (Public Review):
The manuscript by Sidhaye et al. aims to integrate proteomic and transcriptomic analyses of human stem cell-derived cortical brain organoids to identify post-transcriptional regulatory mechanisms during human cortical development. The authors use an innovative and useful dual-reporter strategy to isolate NPCs and neurons separately and integrate proteomic and transcriptomic analyses in each cell type. The data analysis is robust and identifies gene modules with cell class specificity.
While there is no large overlap between the proteomic and transcriptomic datasets, the authors focus additional experiments on one candidate pathway, mTOR-mediated regulation of translation in progenitors, and validate this pathway's role in progenitor development.
The authors also identified a stress-related role for processes …
Reviewer #3 (Public Review):
The manuscript by Sidhaye et al. aims to integrate proteomic and transcriptomic analyses of human stem cell-derived cortical brain organoids to identify post-transcriptional regulatory mechanisms during human cortical development. The authors use an innovative and useful dual-reporter strategy to isolate NPCs and neurons separately and integrate proteomic and transcriptomic analyses in each cell type. The data analysis is robust and identifies gene modules with cell class specificity.
While there is no large overlap between the proteomic and transcriptomic datasets, the authors focus additional experiments on one candidate pathway, mTOR-mediated regulation of translation in progenitors, and validate this pathway's role in progenitor development.
The authors also identified a stress-related role for processes in corticogenesis, although, without comparison to human tissue, it's possible that some of the results are due to the artificial nature of the organoids as they have been reported to have elevated stress (Bhaduri et al.,).
The data is from organoids from one human stem cell line, the female H9 human embryonic stem cell line and so it is critical to validate the results on 1-2 additional stem cell lines, to rule out the possibility that these results are unique to this one cell line.
The major concerns in this paper can be addressed through validation of the results in other systems (e.g. human tissue) or in additional cell lines.
The results provide a valuable resource and address some of the limitations of current organoid and tissue single-cell data by focusing on proteomics.
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