Programmable RNA-binding protein composed of repeats of a single modular unit
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 for...
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| Format: | Article |
| Language: | en_US |
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National Academy of Sciences (U.S.)
2017
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| Online Access: | http://hdl.handle.net/1721.1/109966 https://orcid.org/0000-0003-0547-8129 https://orcid.org/0000-0002-0419-3351 |
| _version_ | 1826217261595099136 |
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| author | Adamala, Katarzyna Martin Alarcon, Daniel Alberto Boyden, Edward |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Adamala, Katarzyna Martin Alarcon, Daniel Alberto Boyden, Edward |
| author_sort | Adamala, Katarzyna |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T17:00:34Z |
| format | Article |
| id | mit-1721.1/109966 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T17:00:34Z |
| publishDate | 2017 |
| publisher | National Academy of Sciences (U.S.) |
| record_format | dspace |
| spelling | mit-1721.1/1099662022-10-03T09:48:23Z Programmable RNA-binding protein composed of repeats of a single modular unit Adamala, Katarzyna Martin Alarcon, Daniel Alberto Boyden, Edward Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences Massachusetts Institute of Technology. Media Laboratory Adamala, Katarzyna Martin Alarcon, Daniel Alberto Boyden, Edward 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. United States. National Institutes of Health (1R01NS075421) National Science Foundation (U.S.) (1344219) United States. National Institutes of Health (1U01MH106011) United States. National Institutes of Health (1R01MH103910) United States. National Institutes of Health (1DP1NS087724) 2017-06-16T18:15:39Z 2017-06-16T18:15:39Z 2016-04 2015-09 Article http://purl.org/eprint/type/JournalArticle 0027-8424 1091-6490 http://hdl.handle.net/1721.1/109966 Adamala, Katarzyna P.; Martin-Alarcon, Daniel A. and Boyden, Edward S. “Programmable RNA-Binding Protein Composed of Repeats of a Single Modular Unit.” Proceedings of the National Academy of Sciences 113, no. 19 (April 2016): E2579–E2588 © 2016 National Academy of Sciences https://orcid.org/0000-0003-0547-8129 https://orcid.org/0000-0002-0419-3351 en_US http://dx.doi.org/10.1073/pnas.1519368113 Proceedings of the National Academy of Sciences Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf National Academy of Sciences (U.S.) PNAS |
| spellingShingle | Adamala, Katarzyna Martin Alarcon, Daniel Alberto Boyden, Edward Programmable RNA-binding protein composed of repeats of a single modular unit |
| title | Programmable RNA-binding protein composed of repeats of a single modular unit |
| title_full | Programmable RNA-binding protein composed of repeats of a single modular unit |
| title_fullStr | Programmable RNA-binding protein composed of repeats of a single modular unit |
| title_full_unstemmed | Programmable RNA-binding protein composed of repeats of a single modular unit |
| title_short | Programmable RNA-binding protein composed of repeats of a single modular unit |
| title_sort | programmable rna binding protein composed of repeats of a single modular unit |
| url | http://hdl.handle.net/1721.1/109966 https://orcid.org/0000-0003-0547-8129 https://orcid.org/0000-0002-0419-3351 |
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