Understanding developmentally controlled co-transcriptional splicing in the mammalian nervous system

Genes are encoded in the form of DNA, a long biopolymer composed of ordered nucleotide units. Cells can retrieve their genetic information by transcribing DNA into RNA copies, which in turn carry instructions for protein synthesis. Before a newly transcribed RNA becomes a fully qualified protein-coding messenger, parts of its sequence known as introns are removed by specialized splicing machinery. Many introns can be excised from nascent RNAs that are still being transcribed from a DNA template. Such co-transcriptional splicing events are thought to be critical for efficient production of correctly formed RNA messengers. It is also possible that regulation of these events during development contributes to global changes in the assortment of RNAs and proteins expressed by the cell. However, molecular mechanisms and functional consequences of developmentally controlled co-transcriptional splicing remain poorly understood.

Our preliminary studies show that hundreds of introns require an RNA-binding protein called Ptbp1 for their efficient co-transcriptional excision. When the cellular pool of Ptbp1 is depleted, some regulated introns become permanently retained in the RNA sequence. This may dampen the expression of protein-coding messengers and give rise to relatively unstable RNA products. We detected the strongest dependence on Ptbp1-activated co-transcriptional splicing for the Dnmt3b gene encoding an important DNA regulator essential for normal embryonic development. Dnmt3b has been associated with devastating medical conditions including cancer, Alzheimer and Parkinson's diseases, mental retardation and immunodeficiencies often leading to life-threatening respiratory infections. Notably, Ptbp1, Dnmt3b and several other co-transcriptionally regulated genes are expressed at relatively high levels in embryonic and/or neural stem cells and progressively downregulated in developing neurons.

With this in mind, we propose to test the hypothesis that Ptbp1 is a key regulator of co-transcriptional splicing and the decline in its abundance in developing neurons facilitates major changes in the excision of introns from nascent RNAs. We will also explore an exciting possibility that this regulation facilitates neuronal differentiation by altering expression of important target genes. We will pursue three interrelated objectives: (1) dissecting molecular mechanisms that allow Ptbp1 to activate co-transcriptional excision of introns; (2) elucidating the effect of co-transcriptional splicing on the abundance, isoform composition, and biological functions of Ptbp1 targets; and (3) understanding the role of Ptbp1 in co-transcriptional splicing dynamics in developing neurons. Our experimental approaches will include monitoring splicing efficiencies in natural and recombinant RNA transcripts, gene editing, neuronal differentiation of embryonic stem cells in vitro, work with primary neurons, and the use of cutting-edge sequencing technologies and advanced bioinformatics tools.

Excision of introns from nascent pre-mRNAs has been proposed to control alternative splicing and the abundance of correctly processed transcripts. How developmental changes in co-transcriptional splicing patterns might contribute to rewiring of the gene expression program is an exciting open question. Building on our preliminary data we will test the hypothesis that the RNA-binding protein Ptbp1 promotes co-transcriptional excision of numerous introns and that its natural downregulation during neuronal differentiation facilitates a large-scale switch between co- and post-transcriptional splicing modes. We will also explore the possibility that this switch facilitates neuronal differentiation by altering expression of important target genes. We will pursue three interrelated objectives: (1) dissecting molecular mechanisms that allow Ptbp1 to activate co-transcriptional excision of introns; (2) elucidating the effect of co-transcriptional splicing on the abundance, isoform composition, and biological functions of Ptbp1 targets; and (3) understanding the role of Ptbp1 in co-transcriptional splicing dynamics in developing neurons. The first two objectives will involve in-depth analyses of Ptbp1-regulated candidates using in vitro and minigene-based assays, auxin-inducible depletion of Ptbp1, and differentiation of genetically modified cells into neurons. Our main model will be the Dnmt3b gene encoding a DNA methylase associated with cancer, immunodeficiency, developmental disorders and neurodegeneration. The third objective will combine unbiased sequencing approaches with viral vector-based Ptbp1 rescue experiments to address the extent to which this protein contributes to co-/post-transcriptional splicing transitions in developing neurons. Overall, this will uncover fundamental mechanisms linking pre-mRNA splicing and gene regulation in developing brain and delineate new possibilities for diagnosing and treating increasingly prevalent medical conditions.