Poster Session 6, 4:15 PM - 5:00 PM: Room 165 [D7]

Developing a Tetracycline-regulated MS2-Tet Aptamer for the Temporal Control of RNA-RBP Interactions

Presenter: Jake William Rosen

Faculty Sponsor: Jiahui (Chris) Wu

School: UMass Amherst

Research Area: Biology

ABSTRACT

RNA–binding proteins (RBPs) are major regulators of RNA metabolism. Upon binding to RNA, RBPs influence RNA stability, translation, splicing, localization, and their assemblies. Conventional approach to control RNA function typically relies on tethering exogenously expressed RBPs to reporter RNAs and measuring resulting phenotypic changes. However, this strategy is limited by overexpression artifacts of RBPs, which can activate cellular stress responses and disrupt endogenous RBP function. In addition, the constitutive nature of these interactions eliminates temporal control, making it difficult to study the dynamic and transient interaction of RNA–RBP in living cells. To overcome these limitations, we recently developed an RNA–regulated destabilization domain system (MS2–mDeg) that selectively stabilizes RBPs only when bound to MS2–containing RNAs, thereby minimizing overexpression artifacts. Despite this advantage, the MS2–mDeg system lacks temporal control, as RNA–RBP interactions remain constitutive. To overcome this limitation, we are developing a small–molecule–regulated platform that enables temporal control of RNA–RBP interactions and RNA metabolism. In this design, an unfolded MS2 aptamer is fused to the P1 stem of a tetracycline–binding RNA aptamer, and RBPs are recruited via mDeg tag. In the absence of tetracycline, the mDeg–tagged RBP undergoes rapid proteasomal degradation, preventing RBP accumulation. However, upon tetracycline addition, the tetracycline–binding aptamer folds, triggering an allosteric conformational change that promotes proper folding of the MS2 aptamer. This folded MS2 aptamer then binds and stabilizes the mDeg–tagged RBP. Together, this platform enables precise, small–molecule regulation of RNA–RBP interactions and provides a powerful tool for studying RNA fate and function in gene expression and disease contexts.