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.

Single-cell transcriptional dynamics in a living vertebrate

“…The her1 -MS2 construct was built off of previously established fusion protein reporter constructs, with the fluorescent protein switched for the MS2 stem loops (Soroldoni et al 2014). Our approach differs from previous works on the MS2 system in zebrafish, which used transient injection of MS2 loops (Campbell et al 2015; Fedder-Semmes and Appel 2021; Costa et al 2020; Zhang et al 2020).…”

“…Given its established genetic toolkit and amenability to live imaging, the zebrafish is a prime candidate for exploring single-cell transcriptional dynamics in vertebrates. While MS2 has been used in zebrafish to study mRNA localization, previous implementations have faced issues of MCP aggregation that prevented quantification of transcription (Campbell et al 2015; Fedder-Semmes and Appel 2021; Costa et al 2020; Zhang et al 2020).…”