Mini-III RNase-based dual-color system for in vivo mRNA tracking

Abstract

Mini-III RNase (mR3), a member of RNase III endonuclease family, can bind to and cleave double-stranded RNAs (dsRNAs). Inactive mR3 protein without the α5β-α6 loop loses the dsRNA cleavage activity, but retains dsRNA binding activity. Here, we establish an inactive mR3-based, non-engineered mR3/dsRNA system for RNA tracking in zebrafish embryos. In vitro binding experiments show that, inactive Staphylococcus epidermidis mR3 (dSmR3) protein possesses the highest binding affinity with dsRNAs among mR3s from other related species, and its binding property is retained in zebrafish embryos. Combined with a fluorescein-labeled antisense RNA probe recognizing the target mRNAs, dSmR3 tagged with an NLS and a fluorescent protein could allow visualizing the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach to track non-engineered RNAs in real-time, which would help understand how endogenous RNAs dynamically move during embryonic development.