UCLA

Pluripotent stem cells form all tissues during development and tissue stem cells are our body’s raw material that can be guided into becoming specialized (differentiated) cells that can be used to regenerate and repair damaged tissues in humans. Alternative Splicing (AS) plays an important role in mediating the switch from a pluripotent stem cell (such as embryonic stem cell (ESC)) to differentiated cell lineages. Switches in mRNA isoforms are mediated by RNA-binding proteins (RBPs), that bind to pre-mRNAs and regulate the inclusion or exclusion of exons in the final mRNA to allow expression of lineage-specific isoforms. In our work, utilizing a unique system for acute depletion of RBPs in mouse ESCs, we have uncovered a new RBP that mediates mRNA isoform switches – Matrin-3 (MATR3). We adopted the auxin-based degron system to investigate the short-term vs long-term effects on splicing and gene expression resulting from loss of the splicing regulator MATR3 in mouse ESCs. Through CRISPR/Cas9-based genome editing, we integrated the auxin-inducible degron tag into the Matr3 loci of mouse ESC lines carrying the TIR ubiquitin ligase, to enable the rapid and conditional degradation of MATR3 protein. We observed that MATR3 is depleted within 1h after addition of auxin, providing an unique tool to study its function in ESCs. We performed RNA-seq across a time course to identify splicing changes from 4h to 48h after auxin addition. We identified several alternative exons that are differently spliced depending on the timing of their knockdown. We then defined a subset of these exons as directly regulated by MATR3 by overlapping crosslinking-immunoprecipitation (iCLIP-seq). Among these targets, we found that MATR3 represses the splicing of exon9 of Csnk1d (Casein kinase 1d), leading to the expression of the isoform Csnk1d2 in ESCs. We find that Csnk1d2 is the dominantly expressed in MEFs and MATR3 regulates this isoform switch. We find the reduction in MATR3 levels pushes the cells towards a more differentiated state, earmarked by both gene expression and splicing changes. Our results point towards a possible role for MATR3 in the maintenance of pluripotency and its effect on differentiation.