Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells
RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-11-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/11/22/3529 |
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| author | Yusuke Yagi Takamasa Teramoto Shuji Kaieda Takayoshi Imai Tadamasa Sasaki Maiko Yagi Nana Maekawa Takahiro Nakamura |
| author_facet | Yusuke Yagi Takamasa Teramoto Shuji Kaieda Takayoshi Imai Tadamasa Sasaki Maiko Yagi Nana Maekawa Takahiro Nakamura |
| author_sort | Yusuke Yagi |
| collection | DOAJ |
| description | RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous <i>CHK1</i> mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics. |
| first_indexed | 2024-03-09T18:25:11Z |
| format | Article |
| id | doaj.art-2a328fe6eeca46a58a0780d41623349a |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-09T18:25:11Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-2a328fe6eeca46a58a0780d41623349a2023-11-24T07:56:55ZengMDPI AGCells2073-44092022-11-011122352910.3390/cells11223529Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian CellsYusuke Yagi0Takamasa Teramoto1Shuji Kaieda2Takayoshi Imai3Tadamasa Sasaki4Maiko Yagi5Nana Maekawa6Takahiro Nakamura7EditForce, Inc., Fukuoka 819-0395, JapanFaculty of Agriculture, Kyushu University, Fukuoka 812-8581, JapanEditForce, Inc., Fukuoka 819-0395, JapanEditForce, Inc., Fukuoka 819-0395, JapanEditForce, Inc., Fukuoka 819-0395, JapanEditForce, Inc., Fukuoka 819-0395, JapanEditForce, Inc., Fukuoka 819-0395, JapanEditForce, Inc., Fukuoka 819-0395, JapanRNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous <i>CHK1</i> mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.https://www.mdpi.com/2073-4409/11/22/3529RNApentatricopeptide repeat proteinsplicing |
| spellingShingle | Yusuke Yagi Takamasa Teramoto Shuji Kaieda Takayoshi Imai Tadamasa Sasaki Maiko Yagi Nana Maekawa Takahiro Nakamura Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells Cells RNA pentatricopeptide repeat protein splicing |
| title | Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells |
| title_full | Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells |
| title_fullStr | Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells |
| title_full_unstemmed | Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells |
| title_short | Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells |
| title_sort | construction of a versatile programmable rna binding protein using designer ppr proteins and its application for splicing control in mammalian cells |
| topic | RNA pentatricopeptide repeat protein splicing |
| url | https://www.mdpi.com/2073-4409/11/22/3529 |
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