Functional impact of alternative splicing coupled to nonsense-mediated decay in developing neurons

Periodic Reporting for period 1 - NEURO_NMD (Functional impact of alternative splicing coupled to nonsense-mediated decay in developing neurons)

Okres sprawozdawczy: 2017-05-01 do 2019-04-30

Embryonic stem cells (ESCs) give rise to all cell types in multicellular eukaryotes. Since cells in the same organism typically share virtually invariant genomic sequences, distinct ESC differentiation outcomes require different sets of genes to be turned on and off in a highly regulated manner. A growing body of evidence points to the importance of post-transcriptional mechanisms in this process. This is particularly evident during development of mammalian neurons, where changes in RNA splicing and stability appear to control gene expression on a truly global scale.. Many important aspects of post-transcriptional regulation in general and a crosstalk between alternative splicing (AS) and RNA destabilization mechanism known as nonsense-mediated decay (NMD) in particular remain poorly understood.
This project aimed to uncover post-transcriptional mechanisms orchestrating neuronal differentiation, with two main technical objectives:
1) Understanding how AS-NMD regulation of newly identified Ptbp1 targets contributes to neuronal development and function.
2) Understanding how NMD target repertoire changes in developing neurons using unbiased time-resolved approaches.
In order to accomplish the objectives of the action, we combined advanced genetic, biochemical, high-throughput and bioinformatics techniques and obtained two main results. 1) We showed that a subset of genes encoding regulators and components of the actin cytoskeleton are co-ordinately downregulated during neuronal differentiation through Ptbp1-dependent AS-NMD. 2) We discovered a novel and unexpected role for Ptbp1 in facilitating co-transcriptional intron removal in a large group of genes expressed in mouse ESCs.
The RNA binding protein Ptbp1 coordinates a large number of key alternative splicing events during early neuronal development. Deregulation of many RNA-based processes has been linked to neurodevelopmental and neurodegenerative diseases. Defects in the NMD machinery itself are known to lead to mental retardation and severe psychiatric conditions including autism, attention deficit hyperactivity disorder and schizophrenia. This project unveiled new important molecular regulation processes in stem cell state maintenance and neuronal development and thus improved our understanding of molecular mechanisms underlying the onset of these serious conditions. Moreover, we produced novel data and tools that should become important resources for the wide scientific community.

The work on this project started with the analysis of previously published data. I then developed and performed experiments to test our initial hypothesis and used the results obtained to extend and refine the scope of our study. Briefly, I analysed transcriptome-wide changes in mouse neuroblastoma cells treated with siRNAs against Ptbp1, and/or the translational inhibitor cycloheximide (CHX; also known to repress NMD) and identified a subset of novel Ptbp1-regulated AS-NMD targets encoding known actin cytoskeleton assembly factors. Analysis of longitudinal RNA-Seq data of in vitro differentiated mouse ESCs into glutamatergic neurons (Hubbard K.S. et al., 2013) confirmed that these targets are naturally downregulated during neuronal differentiation with kinetics similar to that of Ptbp1. I then performed in vitro differentiation of ESCs into neurons and validated the gene downregulation effect and increased inclusion of NMD-inducing (“poison”) exons for several targets. I also confirmed that the inclusion of the poison exon was indeed Ptbp1-dependent, and that it had a direct impact on downregulation of these targets at both mRNA and protein levels.
To gain further insights into the extent of Ptbp1 target repertoire, I established a mouse ESC line (mESC-Ptbp1-AID) that allows for rapid degradation (≃4h) of Ptbp1 using an auxin-inducible depletion system (Natsume T. et al, 2016). I performed several high-throughput experiments with this innovative Ptbp1 downregulation system and identified a previously unknown wider role for Ptbp1 in co-transcriptional intron removal. These experiments also indicated a novel mechanism of how this activity might be coupled with regulation of at least a subset of Ptbp1-controlled alternative exons. Interestingly, examples of the newly identified targets included genes required for the maintenance of the stem cell status and, again, normal assembly of the actin cytoskeleton.
All the genome-wide data generated with the mESC-Ptbp1-AID and the cell line itself provide a valuable community resource, since Ptbp1 is a pivotal regulator of alternative splicing and gene expression in a variety of normal and disease-associated biological contexts. Moreover, we have one manuscript ready for submission and another manuscript currently in preparation to be submitted to peer-reviewed journals.
As part of the dissemination of this project, I presented this research data in several international conferences such as the 2018 RNA society meeting in Berkeley, USA, the 2019 RNA society meeting in Krakow, Poland, and the EMBO workshop “RNP network dynamics in development and disease” in Ljubljana, Slovenia. I also joined outreach events such as the ‘British science week’ and the CDN summer school ‘DevNeuro academy’, which allowed me to discuss my research with students interested in undertaking scientific careers.

This work uncovered novel AS-NMD and co-transcriptional splicing-based mechanisms involved in the maintenance of the stem cell state and neuronal differentiation. Moreover, we unveiled a previously unknown wider role for Ptbp1 in co-transcriptional intron removal. We used and developed advanced experimental techniques in order to accomplish the goals of the project, generating an important set of resources and experimental tools for the field.
We validated the role of AS-NMD in actin cytoskeleton regulation through the combined use of antisense oligonucleotides (AONs) and small interfering RNAs (siRNAs) against Ptbp1 in in vitro differentiated ESCs. In parallel, we identified new Ptbp1-target genes important for pluripotency and neuronal commitment. We also generated a mouse embryonic cell line that allows for rapid, direct depletion of Ptbp1 and generated high-throughput data with this model.
To further elucidate NMD target dynamics in developing neurons, ongoing experiments in the Makeyev lab will focus on the depletion of Ptbp1 at different time points of neuronal differentiation. We attempted different approaches to block the NMD pathway in mouse ESCs but could not achieve this goal due to technical issues.
Finally, we generated a large number of genome-wide data (ChIP-seq, mNET-seq and subcellular fractionation followed by RNA sequencing) with the mESC-Ptbp1-AID. The cell line itself provide a valuable resource for research community, since Ptbp1 is a key regulator of alternative splicing and gene expression in health and disease.

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Periodic Reporting for period 1 - NEURO_NMD (Functional impact of alternative splicing coupled to nonsense-mediated decay in developing neurons)

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