Advances in next-generation sequencing have revealed that cancer and neurological disorders are often associated with distinct transcriptomic signatures, including widespread alterations in alternative splicing, a process that vastly expands the coding potential of the genome. Loss- or gain-of-function mutations in RNA binding proteins impact entire networks of coordinated RNA processing events, which play critical roles in cell fate decisions and impact processes such as cell cycle control, neurogenesis and synaptic transmission. However, given that the function of the overwhelming majority of such events, including alternative cassette exons, remains unexplored, Thomas Gonatopoulos-Pournatzis’ research program aims to identify individual events that control these fundamental biological processes, reveal how their disruption contributes to disease- or disorder-related phenotypes, and explore the regulatory networks that control RNA processing.  

1.  Exon-resolution functional genomics

To advance the systematic mapping of genetic interactions and interrogation of the functions of sizeable genomic segments, we have developed a CRISPR-based screening system for combinatorial genetic perturbations, named CHyMErA. CHyMErA employs co-expression of Cas9 and Cas12a nucleases and libraries of hybrid Cas9-Cas12a guide RNAs that can be employed for multi-targeted genome editing. We have applied CHyMErA in human cells to uncover novel genetic interactions between paralogous gene pairs. Importantly, we have also used CHyMErA to systematically target thousands of alternative splicing events, identifying dozens of exons underlying cell fitness. CHyMErA thus represents an effective screening approach for genetic interaction mapping and the functional analysis of sizeable genomic regions, such as alternative exons.

Functional Exomics: Future application of CHyMErA screens coupled to high-throughput phenotyping will enable the efficient functional characterization of alternative exons in the human genome and the identification of novel targets for precision medicine applications using high resolution phenotypic profilling.

Technology Development: A major goal of the Gonatopoulos-Pournatzis team is to continue developing cutting-edge technologies that will enable the systematic and efficient functional characterization of transcript isoform diversity and genetic interactions using phenotypically rich screening readouts.  

Image

2. Mapping gene regulatory networks that control alternative splicing

An important challenge in biomedical research is to elucidate the gene regulatory networks that underlie the establishment of complex transcript isoform expression patterns in a cell-type specific manner, and understand how their disruption contributes to disease-states. We have previously developed a CRISPR-based screening platform that allows the systematic identification of regulatory factors that control alternative splicing. CRISPR-Cas9 loss-of-function genome wide screens in neural cells expressing dual-fluorescent splicing reporters uncovered hundreds of splicing regulators and have shed light on how very short neuronal exons, linked to autism spectrum disorders, are recognized and spliced. The aforementioned strategy represents a highly effective approach for the comprehensive definition of splicing regulatory pathways. Future studies using this tool will systematically delineate regulatory pathways that control the splicing of alternative exons with critical roles in mammalian cells.  

Image

3.  Uncover microexons that impact animal behavior and cognitive functioning

A program of neuronal-specific, highly conserved microexons is frequently disrupted in the brains of patients with autism spectrum disorder (ASD). We have started to identify individual microexons that contribute to ASD-associated phenotypes by characterizing a set of neuronal microexons in the translation initiation factors eIF4G1 and eIF4G3. The eIF4G microexons selectively up-regulate synaptic proteins that control neuronal activity and plasticity, and mice lacking the Eif4g1 microexon display social behavior as well as learning and memory deficits. Future studies are focused on uncovering additional alternative exons with such critical roles for animal behavior and cognitive functioning. 

Image

Diversity and Inclusion Statement 

Our group strives to create and maintain a welcoming and equitable environment. We welcome all qualified individuals regardless of race, color, religion, sex (including pregnancy, sexual orientation, and gender identity), national origin, age, disability, and genetic information.