Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells

Yusuke Yagi, Takamasa Teramoto, Shuji Kaieda, Takayoshi Imai, Tadamasa Sasaki, Maiko Yagi, Nana Maekawa, Takahiro Nakamura

Research output: Contribution to journalArticlepeer-review

Abstract

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 CHK1 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.

Original languageEnglish
Article number3529
JournalCells
Volume11
Issue number22
DOIs
Publication statusPublished - Nov 2022

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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