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Abstract
Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.
Highlights
Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats
Random Library Screen for Cytosine Recognition—To select a PUM repeat that recognizes cytosine, we used a Y3H system that utilizes co-expression of the PUF domain fused with the Gal-4 activation domain, an RNA target with an MS2-binding site, and an MS2-LexA fusion protein (Fig. 1B)
Repeat 5 of wild-type PUF has an arginine (Arg-1008) in position to stack with the RNA base, and we found that the two mutations in the edge-interacting side chains were sufficient for cytosine recognition
Summary
Using a yeast three-hybrid system, we found that the 5-residue RNA interaction sequence SYXXR allows PUM repeats of human Pumilio 1 (hereafter referred to as PUF for simplicity) to interact with cytosine. In a crystal structure of a complex between a mutant PUF (SYXXR) and cognate RNA, the arginine side chain interacts directly with the cytosine, and the serine side chain helps to position the arginine residue. We applied the recognition code to design new PUF domains to recognize RNA targets with multiple cytosine residues such as CUG repeats that are responsible for the pathogenesis of myotonic dystrophy. A naturally occurring yeast PUF protein, Nop9p, appears to contain a repeat with a code for cytosine and is conserved in homologs from yeast to human, suggesting that the natural target sequences of these PUF proteins may contain cytosine
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