Research Overview
Our research focuses on mechanisms underlying the regulation of gene expression and how these mechanisms are disrupted in human diseases and disorders. Most of our research is directed at understanding how alternative splicing is regulated and integrated with other layers of gene expression to control fundamental biological processes. For example, we have discovered alternative splicing "switches" with critical roles in the regulation of transcriptional programs required for neural and embryonic stem cell fate. More recently, we have discovered and characterized a highly conserved alternative splicing regulatory network comprising 3-27 nt neuronal microexons, and have provided evidence that the disruption of this network represents a common mechanism underlying autism spectrum disorders.
Our highly collaborative research program utilizes a wide range of approaches, from bioinformatics and functional genomics to focused molecular, biochemical, cell biological methods, as well as the generation of animal models. We have pioneered the development and application of technologies for the genome-wide quantitative profiling of transcriptomes, RNA interactomes, as well as new CRISPR-based methods designed to comprehensively elucidate RNA regulatory networks. These efforts are uncovering remarkable landscapes of new regulation that await further investigation. They are also providing insight into possible new therapeutic strategies for the treatment of human diseases and disorders.
During the past five years our research has been supported by funding from the following agencies: Canadian Institutes of Health Research, Natural Sciences and Engineering Research Council of Canada, National Institutes of Health, Canadian Research Excellence Fund Medicine by Design Program, Simons Foundation, Canadian Stem Cell Network, and University of Toronto Covid-19 Action Fund.
Selected Ongoing Projects
1. Systematic discovery and functional characterization of splicing regulatory networks
A key goal in biomedical research is to systematically identify and characterize the regulation and function of RNA networks. In collaboration with Jason Moffat, Jeffrey Wrana and colleagues, we have developed new functional genomic tools, including a genome-wide CRISPR Cas9-based approach, enabling the comprehensive identification of trans-acting factors that control biologically important alternative splicing events (Han, Braunschweig et al. Mol Cell, 2017; Gonatopoulos-Pournatzis et al. Mol Cell, 2018). These strategies have revealed entire networks of trans-acting regulators that control neuronal microexons disrupted in autism (see below), as well as important stem cell fate-determining splicing events discovered in our previous research (e.g. Gabut et al. Cell, 2011; Han et al. Nature, 2013). An important current goal is to develop screens enabling the systematic interrogation of the functions of individual exons and other elements comprising splicing and other RNA networks. To this end, together with Jason Moffat’s group, we have developed a new dual Cas-CRISPR system that enables the large-scale interrogation of exon function (Gonatopoulos-Pournatzis et al. Nature Biotech, 2020). Future work will harness this and related approaches to define the functions various splicing regulatory networks with important roles in cell fate, development and disease.
2. Microexons: regulation, function and roles in neurological disorders
We previously discovered a large program of highly conserved and neuronal-specific, 3-27 nucleotide-long ‘microexons’ (Irimia et al. Cell, 2014). A remarkable feature of these short exons is that they are often misregulated in the brains of individuals with autism spectrum disorder. We have linked this altered regulation to the loss of expression of a neuronal-specific splicing regulator (nSR100/SRRM4) that was previously discovered in our lab (Calarco et al. Cell, 2009). In collaboration with Sabine Cordes (LTRI, Mt. Sinai Hospital) and other colleagues in Toronto, we have further shown that mice haploinsufficient of SRRM4 recapitulate microexon disruption and display multiple, hallmark autistic-like features (Quesnel-Vallieres et al. Genes Dev. 2015; Mol Cell, 2016). More recently, we have shown that disruption of a single SRRM4-regulated microexon in the translation initiation factor eIF4G1 alters synaptic protein translation and higher-order cognitive functioning, through a mechanism related to that observed in Fragile X Syndrome, the most common monogenetic form of autism (Gonatopoulos-Pournatzis et al. Mol Cell, 2020). Our current research is directed at developing a deeper understanding of microexon regulation and function, as well as therapeutic strategies for correcting microexon deficiency.
3. Exploring structural roles of RNAs in gene regulation and cell organization
More than half of the human genome is transcribed into RNA. A major challenge in biological research is to determine which of the myriad of transcripts that lack apparent protein-coding capacity provide important functional roles. Similarly, numerous abundant and conserved transcripts that bear hallmarks of previously characterized non-coding RNAs have been identified but also lack defined functions. To address these challenges, we have developed a method for the systematic mapping of RNA-RNA interactions in cells, referred to as ‘LIGR-Seq’ (Sharma et al. Mol Cell, 2016). This method detects RNA-RNA duplexes cross-linked in vivo, through the ligation of proximal free ends, followed by sequencing of the resulting chimeric junctions. LIGR-Seq and a more recent and sensitive version of this protocol has resulted in the detection of hundreds of new RNA-RNA interactions that yield testable hypotheses as to the possible functions of previously uncharacterized non-coding RNAs. In future work we will investigate these new functions, and further apply LIGR-Seq to uncover regulatory landscapes controlled by RNA-RNA interactions during cell fate determination and in the context of human diseases. Extending our previous work (Gueroussov et al. Cell, 2017), we are also applying orthogonal methods to define RNAs that contribute to formation of multivalent ribonucleoprotein assemblies and cellular domains, to gain deeper insight into nuclear structure-function relationships.
4. Elucidation of sequence and structural codes that control RNA regulation
Extending our pioneering work elucidating features of the splicing code (Barash et al. Nature, 2010; Barbosa-Morais et al. Science 2012; Braunschweig et al. Genome Res. 2014), in collaboration with Quaid Morris we are currently striving to decipher a more complete code that accounts for all classes of alternative splicing, as well as additional layers of RNA regulation such as alternative 3´end formation (Ha et al. Genome Biol. 2018). Understanding a more complete RNA code is a key step in determining how genes are regulated and how mutations and other forms of genetic variation cause or contribute to human diseases and disorders.