Abstract
Subcellular localizations of RNAs can be imaged in vivo with genetically encoded reporters consisting of a sequence-specific RNA-binding protein (RBP) fused to a fluorescent protein. Several such reporter systems have been described based on RBPs that recognize RNA stem-loops. Here we describe RNA tagging for imaging with an inactive mutant of the bacterial endonuclease Csy4, which has a significantly higher affinity for its cognate stem-loop than alternative systems. This property allows for sensitive imaging with only few tandem copies of the target stem-loop inserted into the RNA of interest.
Keywords:
Csy4; Filamentous fungi; Live-cell imaging; Neurospora crassa; Plant virus; RNA imaging; RNA stem-loop; RNA virus.
MeSH terms
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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CRISPR-Associated Proteins / genetics*
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CRISPR-Associated Proteins / metabolism
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Cloning, Molecular
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Endoribonucleases / genetics*
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Endoribonucleases / metabolism
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Fungi / genetics*
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Fungi / metabolism
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Gene Expression / genetics
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Genes, Reporter / genetics*
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Inverted Repeat Sequences / genetics
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Microscopy, Confocal / methods*
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Mutation
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Neurospora crassa / genetics
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Neurospora crassa / metabolism
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Nicotiana / genetics
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Nicotiana / metabolism
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Nicotiana / virology
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Plant Leaves / genetics
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Plant Leaves / metabolism
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Plants / genetics*
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Plants / metabolism
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Plants / virology
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Protein Binding
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RNA / genetics*
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RNA / metabolism
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RNA-Binding Proteins / genetics*
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RNA-Binding Proteins / metabolism
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Transformation, Genetic
Substances
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Bacterial Proteins
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CRISPR-Associated Proteins
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Luminescent Proteins
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RNA-Binding Proteins
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RNA
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Csy4 endoribonuclease, Pseudomonas aeruginosa
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Endoribonucleases