Splicing in a single neuron is coordinately controlled by RNA binding proteins and transcription factors
Morgan Thompson, Ryan Bixby, Robert Dalton, Alexa Vandenburg, John A Calarco, Adam D Norris Is a corresponding author
Southern Methodist University, United States; University of Toronto, Canada
RESEARCH ARTICLE Jul 19, 2019
DOI: 10.7554/eLife.46726
Abstract
Single-cell transcriptomes are established by transcription factors (TFs), which determine a cell's gene-expression complement. Post-transcriptional regulation of single-cell transcriptomes, and the RNA binding proteins (RBPs) responsible, are more technically challenging to determine, and combinatorial TF-RBP coordination of single-cell transcriptomes remains unexplored. We used fluorescent reporters to visualize alternative splicing in single Caenorhabditis elegans neurons, identifying complex splicing patterns in the neuronal kinase sad-1. Most neurons express both isoforms, but the ALM mechanosensory neuron expresses only the exon-included isoform, while its developmental sister cell the BDU neuron expresses only the exon-skipped isoform. A cascade of three cell-specific TFs and two RBPs are combinatorially required for sad-1 exon inclusion. Mechanistically, TFs combinatorially ensure expression of RBPs, which interact with sad-1 pre-mRNA. Thus a combinatorial TF-RBP code controls single-neuron sad-1 splicing. Additionally, we find ‘phenotypic convergence,’ previously observed for TFs, also applies to RBPs: different RBP combinations generate similar splicing outcomes in different neurons.
eLife digest
All the cells in the human nervous system contain the same genetic information, and yet there are many kinds of neurons, each with different features and roles in the body. Proteins known as transcription factors help to establish this diversity by switching on different genes in different types of cells.
A mechanism known as RNA splicing, which is regulated by RNA binding proteins, can also provide another layer of regulation. When a gene is switched on, a faithful copy of its sequence is produced in the form of an RNA molecule, which will then be ‘read’ to create a protein. However, the RNA molecules may first be processed to create templates that can differ between cell types: this means that a single gene can code for slightly different proteins, some of them specific to a given cell type. Yet, very little is known about how RNA splicing can generate more diversity in the nervous system.
To investigate, Thompson et al. developed a fluorescent reporter system that helped them track how the RNA of a gene called sad-1 is spliced in individual neurons of the worm Caenorhabditis elegans. This showed that sad-1 was turned on in all neurons, but the particular spliced versions varied widely between different types of nerve cells.
Additional experiments combined old school and cutting-edge genetics technics such as CRISPR/Cas9 to identify the proteins that control the splicing of sad-1 in different kinds of neurons. Despite not directly participating in RNA splicing, a number of transcription factors were shown to be involved. These molecular switches were turning on genes that code for RNA binding proteins differently between types of neurons, which in turn led sad-1 to be spliced according to neuron-specific patterns.
The findings by Thompson et al. could provide some insight into how mammals can establish many types of neurons; however, a technical hurdle stands in the way of this line of research, as it is still difficult to detect splicing in single neurons in these species.
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