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Dynamics and consequences of widespread transcript isoform changes during neuronal differentiation

ABSTRACT: Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform change upon cell-state transitions during cell differentiation, and the determinants and functional consequences have largely remained unclear. Here, we used an improved model for human neurogenesis in vitro that we show is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct stages during neurogenesis. RNA isoforms mainly arise from the alternative usage of transcription start sites and poly-adenylation sites as well as the skipping of individual exons during human neurogenesis. The transcript isoform changes can remodel the abundance and functions of protein isoforms. Finally, our study identifies a set of RNA-binding proteins as a likely determinant of differentiation stage-specific global isoform changes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pluripotent Stem Cell, Bipolar Neuron, Neuronal Stem Cell

SUBMITTER: David Meierhofer

LAB HEAD: Andreas Mayer

PROVIDER: PXD046084 | Pride | 2024-04-11

REPOSITORIES: Pride

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Project description:Dosage imbalance of X-chromosomal genes contributes to sex differences, in particular during early development, when both X chromosomes are active in females. X-encoded inhibitors of the differentiation-promoting MAP kinase (MAPK) signalling pathway slow down development, increase levels of naive pluripotency factors and decrease MAPK target gene expression. Through a hierarchical CRISPR screening approach in murine embryonic stem cells (mESC) we have comprehensively identified X-linked genes that modulate MAPK signalling, pluripotency factor expression and differentiation. We show that multiple genes act in concert to drive sex differences in mESC. Dusp9, a known negative regulator of the MAPK pathway alters phosphorylation of MAPK pathway intermediates in female cells, while Klhl13 underlies differences in pluripotency factor expression and differentiation by targeting differentiation-promoting factors for degradation. Klhl13 is a member of the Cullin 3 E3 ubiquitin ligase complex, where it acts as a substrate adaptor to target proteins for proteasomal degradation (Dhanoa et al., 2013). We reasoned that the Klhl13-mediated sex differences we have identified might be due to reduced protein abundance of Klhl13 substrate proteins in female compared to male cells, which affect pluripotency factors, differentiation and MAPK target gene expression. To identify Klhl13 substrates in mESCs, we comprehensively profiled Klhl13 interaction partners and then selected those with increased protein levels in K13-HOM mutant cells. To identify interaction partners, we ectopically expressed either full-length Klhl13 or the substrate-binding Kelch domain, tagged with a green fluorescent protein (GFP), and identified binding partners by Immunoprecipitation-Mass Spectrometry (IP-MS) using a GFP-specific antibody. These experiments were performed in female mESCs with a homozygous Klhl13 deletion. By using this approach, we identify Scml2 and Alg13 as Klhl13 substrates.

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Dynamics and consequences of widespread transcript isoform changes during neuronal differentiation

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