Poster abstracts

Poster number 37 submitted by Thomas Gallagher

Exploring the function of the EJC and NTC during zebrafish development

Thomas L. Gallagher (Molecular Genetics, The Ohio State University), Kiel T. Tietz (Molecular Genetics, The Ohio State University), Pooja S. Gangras (Molecular Genetics, The Ohio State University), Natalie C. Deans (Molecular Genetics, The Ohio State University), Guramrit Singh (Molecular Genetics, The Ohio State University), Sharon L. Amacher (Molecular Genetics, The Ohio State University)

Abstract:
Components of the exon junction (EJC) and nineteen complexes (NTC) are extraordinarily conserved across developmental phyla. Depletion of these factors in vertebrate systems reveals cell- or tissue-specific defects, despite the presumed ubiquitous role of the NTC for pre-mRNA splicing and the EJC for nuclear export, localization, and/or decay of mRNA. These observations hint at shared and non-overlapping roles for EJC and NTC factors and/or EJC and NTC regulation by cell- or tissue-specific regulators. However, the precise roles of the EJC and NTC in post-transcriptional gene regulation during development and differentiation are largely unknown. To characterize developmental and tissue-specific roles of the EJC and NTC, we have isolated zebrafish mutants of core components of each complex.

Using genome-wide SNP association mapping, we molecularly identified the honu lesion as a nonsense mutation in the splicing factor Cdc5l. Cdc5l is a core component of the NTC and plays an important role in pre-mRNA splicing and mitotic progression in mammalian and yeast cells. In zebrafish, honu mutants do not complete segmentation and accumulate cyclic transcripts that are normally cleared rapidly during segmentation. This accumulation occurs post-splicing since expression of nascent transcripts appears normal in these mutants. High resolution detection of cyclic transcripts in honu mutants reveals a pronounced nuclear accumulation defect, suggesting that mRNA export may be compromised. Since late stage spliceosomal components like the NTC interface with the EJC in mammalian cells, we are now exploring the possibility that EJC mutants share similar defects in mRNA stability and localization with honu mutants. Using CRISPR/Cas9 technology, we have generated null alleles in the core EJC genes magoh, eif4a3, and y14/rbm8a. Phenotypic characterization is underway, in addition to global transcript profiling by RNA-seq of wild-type and mutant embryos. In parallel, we are exploring the potential impact that mutation of EJC and NTC core factors has on nonsense mediated decay (NMD) by quantifying Upf1-regulated NMD candidates in EJC and NTC mutants.

Keywords: EJC, NTC, Cdc5l