An inducible tethered function assay reveals differential regulatory effects for the RNA-binding protein YBX3 and its domains.

Abstract

RNA-binding protein (RBP) regulation is widespread across biology from development to learning and memory. Often RBPs contain multiple modular domains, which contribute to distinct RNA- binding activity or interactions necessary for regulation. However, assays to determine specific regulatory activity of individual domains are limited. The tethered function assay (TFA) provides a direct method to assess the functional activity of RBPs and their domains. The assay consists of two key components: (i) a reporter plasmid that encodes an mRNA, such as luciferase or GFP, engineered with high-affinity binding sites for an exogenous RNA-binding factor, and (ii) an effector construct that expresses a chimera of the RBP fused to the RNA-binding factor. Co- transfection of these constructs allows for measurement of reporter activity as a quantitative readout for RBP regulatory function. We developed an inducible TFA (iTFA) system through generation of a stable inducible mammalian cell line to express a reporter mRNA encoding nanoluciferase (nLuc) with six high-affinity MS2 binding sites within its 3 untranslated region. These cells can be transfected with a single plasmid that expresses an RBP fused to the MS2 coat protein. This approach enhances efficiency and reproducibility through reduction of transfection burden to a single plasmid and simplified normalization. We used this platform to dissect the individual and combined regulatory effects of YBX3 domains, a modular RBP with differential regulatory activity. The iTFA system provides a streamlined, tunable platform for functional analysis of RBPs that facilitates rapid interrogation of RBP or domain-specific activities in mammalian cells.