Open AccessMini-III RNase-based dual-color system for in vivo mRNA tracking
Author(s) -
Lin Zhang,
Luxi Chen,
Jing Chen,
Wei-Min Shen,
Anming Meng
Publication year - 2020
Publication title -
development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.15
H-Index - 36
eISSN - 1477-9129
pISSN - 0950-1991
DOI - 10.1242/dev.190728
Subject(s) - biology , rna silencing , rnase p , rna , zebrafish , rnase h , ribonuclease iii , microbiology and biotechnology , messenger rna , rna binding protein , endonuclease , biochemistry , gene , rna interference
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 a nuclear localization sequence and a fluorescent protein could allow visualization of the dynamics of endogenous target mRNAs. The dSmR3/antisense probe dual-color system provides a new approach for tracking non-engineered RNAs in real-time, which will help understand how endogenous RNAs dynamically move during embryonic development.