Targeted translational regulation using the PUF protein family scaffold

Abstract

Regulatory complexes formed on mRNAs control translation, stability, and localization. These complexes possess two activities: one that binds RNA and another-the effector-that elicits a biological function. The Pumilio and FBF (PUF) protein family of RNA binding proteins provides a versatile scaffold to design and select proteins with new specificities. Here, the PUF scaffold is used to target translational activation and repression of specific mRNAs, and to induce specific poly(A) addition and removal. To do so, we linked PUF scaffold proteins to a translational activator, GLD2, or a translational repressor, CAF1. The chimeric proteins activate or repress the targeted mRNAs in Xenopus oocytes, and elicit poly(A) addition or removal. The magnitude of translational control relates directly to the affinity of the RNA-protein complex over a 100-fold range of K d . The chimeric proteins act on both reporter and endogenous mRNAs: an mRNA that normally is deadenylated during oocyte maturation instead receives poly(A) in the presence of an appropriate chimera. The PUF-effector strategy enables the design of proteins that affect translation and stability of specific mRNAs in vivo.CAF1/RNA stability | GLD2/polyadenylation

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

“…By linking a modified PUF to an effector domain, regulation can be targeted to specific mRNAs (39)(40)(41). The plasticity of interactions reported here raises considerations for such studies: Flexibility may cause off-target effects.…”

An artificial PPR scaffold for programmable RNA recognition

“…We showed that our individual PPR modules can be combined within the engineered cPPR proteins with the capacity to modify their binding specificities to single-nucleotide level, providing further evidence of their modularity. We thus demonstrate that they might be engineered to target and manipulate RNAs of interest, as has been the case for PUFs 7,26,37,[39][40][41][42][43][44][45][46] . Here we focused on engineered proteins with eight PPRs.…”

The Nucleic Acid-binding Domain and Translational Repression Activity of a Xenopus Terminal Uridylyl Transferase

“…Reporter RNA Plasmids-The pLG-MS2 (firefly luciferase), pLG-MS2ϩA39 (polyadenylated firefly luciferase), pSP65-ren (Renilla luciferase), pLGMS2-luc (RNA with three MS2-binding sites), pLGMS2ϩA39-luc (RNA with three MS2-binding sites and a poly(A) 39 tail), pLG:FBE-ACAmut (RNA that lacked MS2 binding sites), and pLG:FBE-ACAmutϩA39 (RNA with a poly(A) 39 tail that lacked MS2 binding sites) plasmids have been described (41)(42)(43)(44).…”

“…Luciferase Assays and Western Blotting-Dual-Luciferase assays (Promega) and Western blotting were performed as described (35,43). Student's two-tailed t tests were used to calculate all p values.…”