Sequence-specific targeting of RNA

Abstract

Post-transcriptional modifications play an important role in several processes, including translation, splicing, and RNA degradation in eukaryotic cells. To investigate the function of specific modifications it is of high interest to develop tools for sequence-specific RNA-targeting. This work focuses on two abundant modifications of eukaryotic mRNA, namely methylation of the guanine-N7 position of the 5'-cap and internal N⁶-methyladenosine (m⁶A). We describe the sequence-specific targeting of model RNA transcripts via RNA-binding proteins, such as nuclease-deficient RNA-targeting Cas9 (RCas9) and the Pumilio homology domain (PumHD) fused to two different effector enzymes, the dioxygenase FTO and the guanine-N7 methyltransferase Ecm1. With this tool, we were able to install and remove the methylation at the respective positions with high specificity.

Keywords: Ecm1; FTO; Methyltransferase; N(6)-methyladenosine (m(6)A); RNA modification; RNA-binding proteins.