RNA-binding proteins (RBPs) serve as a key player in the post-transcriptional gene regulation, including alternative pre-mRNA splicing (AS). Despite their significant role in cellular function, most of the over 2,000 human proteins predicted or shown to bind RNA lack an assigned molecular function. AS is a highly important RNA processing step, with up to 95% of human multi-exon genes exhibiting multiple splice isoforms. Aberrant splicing is prevalent in disease, particularly cancer, leading to proteomic imbalance and disruption of cellular homeostasis. RBPs involved in AS lack functional annotation of their RNA-binding activity, necessitating systematic approaches to bridge this knowledge gap.
Previous assays have relied on global overexpression or knockdown of RBPs to identify and characterize RBPs that underscore AS. However, global perturbations of protein level do not allow separation of effects caused by direct binding of RBPs from their indirect action through splicing regulatory networks. Our study developed tethered function luciferase-based splicing reporter assays to investigate and quantify the protein sequence’s capacity to directly promote exon inclusion. We used this system to systematically assess proximity-dependent modulation of exon inclusion for 718 human RBPs at two separate tethering positions and to identify potent and compact exon inclusion activation domains.
We constructed dual-luciferase tethered AS minigene reporter systems based on the splicing event of MAPT (microtubule-associated protein tau) exon 10, which is predominantly excluded from the mature mRNA in HEK293T cells. The first reporter contains the MS2 hairpin 30 base pairs downstream of the 5′ splice site (lucMAPT-30D), and the second contains the MS2 hairpin 30 base pairs upstream of the 3′ splice site (lucMAPT-30U).
To read more about this study, click here: Read More.
