The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby-RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.
|Original language||English (US)|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - May 10 2016|
Bibliographical noteFunding Information:
We thank Katriona Guthrie-Honea and Paul Reginato for assistance with cloning and Kiryl Piatkevich, Jacob Becraft, Daniel Schmidt, and Stuart Levine for advice. This work was supported by NIH Grant 1R01NS075421, National Science Foundation Chemical, Bioengineering, Environmental, and Transport Systems Grant 1344219, Jeremy and Joyce Wertheimer, NIH Brain Research through Advancing Innovative Neurotechnologies Initiative Grant 1U01MH106011, the New York Stem Cell Foundation Robertson Award, NIH Director's Transformative Award 1R01MH103910, and NIH Director's Pioneer Award1DP1NS087724 (to E.S.B.), the Janet and Sheldon Razin (1959) Fellowship (to D.A.M.-A.), and the Massachusetts Institute of Technology Media Lab.
- Gene expression monitoring
- Protein engineering
- RNA-binding protein
- Translation initiation