Characterization of Functional Domains of CASC3 in mRNA Nuclear Export

Abstract

CASC3 （also known as BTZ or MLN51） is a peripheral component of the exon junction complex （EJC）. The EJC facilitates the recruitment of the TREX complex and other factors involved in mRNA nuclear export. Previous work from our laboratory showed that CASC3 depletion causes the nuclear accumulation of poly（A）+ RNA, nascent RNA, and intronless RNA, suggesting an important role for CASC3 in mRNA export. This study investigated the functional domains of CASC3 required for this activity and characterized its RNA-binding profile. Six CASC3 domain-deletion mutants were generated by site-directed mutagenesis and expressed in HeLa cells. Deletion of the nuclear export signal （NES） did not substantially impair CASC3-induced nuclear accumulation of poly（A）+ RNA, indicating that the NES is dispensable for this activity. In contrast, deletion of the RNA-binding motif （RBM） or nuclear localization signal （NLS） regions altered poly（A）+ RNA distribution. Rescue experiments in CASC3-depleted cells further showed that RBM- or NLS-deficient mutants failed to mRNA exprot. To characterize the RNA-binding properties of CASC3, published iCLIP datasets for CASC3, the mRNA export receptor NXF1, the mRNA export adaptor ALYREF, and the EJC component eIF4A3 were analyzed. CASC3’s binding profile was more similar to that of ALYREF than to those of eIF4A3 or NXF1. Functional enrichment analysis of CASC3-enriched targets identified RNA processing and mRNA transport related pathways, whereas motif analysis revealed little exact sequence overlap among the four proteins.