Abstract
BackgroundDue to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. To further facilitate the application of the PUF domain for the sequence-specific RBP engineering, a rapid cloning approach is desirable that would allow efficient introduction of multiple key amino acid mutations in the protein. Here, we report the implementation of the Golden Gate cloning method for an efficient one-step assembly of a designer PUF domain for RNA specificity engineering.ResultsWe created a repeat module library that is potentially capable of generating a PUF domain with any desired specificity. PUF domains with multiple repeat modifications for the recognition of altered RNA targets were obtained in a one-step assembly reaction, which was found to be highly efficient. The new PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular approach for PUF engineering. To demonstrate the application of the PUF domain assembly method for RBP engineering, we fused the PUF domain to a post-transcriptional regulator and observed a sequence-specific reporter and endogenous gene repression in human cell lines.ConclusionsThe Golden Gate based cloning approach thus should allow greater flexibility and speed in implementing the PUF protein scaffold for engineering designer RBPs, and facilitate its use as a tool in basic and applied biology and medicine.
Highlights
Due to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering
We report the implementation of the Golden Gate (GG) cloning, Type IIS restriction endonuclease-based approach [29] for engineering of PUF-based RBPs
As a scaffold for the development of the assembly toolkit, we used the human Pumilio homology domain (PUM-HD), which consists of the amino acids 828-1176 of the full-length Pumilio 1 (PUM1) [21]
Summary
Due to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. Hundreds to thousands of eukaryotic proteins are estimated to function as RBPs [1] and govern many aspects of RNA biology including translation, turnover, processing, and cellular localization [2,3,4]. Despite their great diversity in function, only a few types of RNA-binding domains are known, which are combined in different structural arrangements with a variety of functional domains [5]. Only pentatricopeptide repeat [13,14] and Pumilio/ fem-3 mRNA binding factor (PUF) [15,16,17,18] proteins have been demonstrated to have the potential to be rationally modified for predictable and specific RNA recognition
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