Fluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells...
moreFluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells and having desirable photophysical properties. Our goal was to create a variant of DsRed-Express that maintains the brightness, fast-maturation, and photostability of this protein,
The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of...
moreThe ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.
Tools to study the abundance, distribution, and flux of intracellular molecules are crucial for understanding cellular signaling and physiology. Although powerful, the current FRET-based technology for imaging cellular metabolites is not...
moreTools to study the abundance, distribution, and flux of intracellular molecules are crucial for understanding cellular signaling and physiology. Although powerful, the current FRET-based technology for imaging cellular metabolites is not easily generalizable. Thus, new platforms for generating genetically encoded sensors are needed. We recently developed a new class of biosensors on the basis of Spinach, an RNA mimic of GFP. In this case, RNA aptamers against a target ligand are modularly fused to Spinach that substantially induce Spinach fluorescence in the presence of ligand. We have used this approach to detect metabolites and proteins both in vitro and in living bacteria, thus providing an alternative to FRET-based sensors and a generalizable approach for generating fluorescent sensors to any ligand of interest.
RNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, Fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a non-coding r(CGG) repeat expansion...
moreRNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, Fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a non-coding r(CGG) repeat expansion (r(CGG)exp) that: (i) sequesters proteins involved in RNA metabolism in nuclear foci, causing deregulation of alternative pre-mRNA splicing; and (ii) undergoes repeat associated non-ATG translation (RANT), which produces toxic homopolymeric proteins without using a start codon. Here we describe the design of two small molecules that inhibit both modes of toxicity and the implementation of various tools to study perturbation of these cellular events. Competitive Chemical Cross Linking and Isolation by Pull Down (C-Chem-CLIP) established that compounds bind r(CGG)exp in cells and also defined small molecule occupancy of RNA targets in cells, the first approach to do so. Using an RNA GFP mimic, r(CGG)exp-Spinach2, we observe that our optimal designed compound binds r(CGG)exp and affects RNA localization by disrupting pre-formed RNA foci. These events correlate with improvement of pre-mRNA splicing defects caused by RNA gain-of-function. In addition, the compounds reduced the formation of toxic homopolymeric proteins formed via RANT. Polysome profiling studies showed that small molecules decreased loading of polysomes onto r(CGG)exp, explaining decreased translation.
In mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell,...
moreIn mammalian cells under oxidative stress, the methionyl-tRNA synthetase (MetRS) misacylates noncognate tRNAs at frequencies as high as 10% distributed among up to 28 tRNA species. Instead of being detrimental for the cell, misincorporation of methionine residues in the proteome reduces the risk of oxidative damage to proteins, which aids the oxidative stress response. tRNA microarrays have been essential for the detection of the full pattern of misacylated tRNAs, but have limited capacity to investigate the misacylation and mistranslation mechanisms in live cells. Here we develop a dual-fluorescence reporter to specifically measure methionine misincorporation at glutamic acid codons GAA and GAG via tRNA(Glu) mismethionylation in human cells. Our method relies on mutating a specific Met codon in the active site of the fluorescent protein mCherry to a Glu codon that renders mCherry nonfluorescent when translation follows the genetic code. Mistranslation utilizing mismethionylated tRN...
Fluorescent proteins (FPs) are invaluable tools for biomedical research. Useful FPs have desirable fluorescence properties such as brightness and photostability, but a limitation is that many orange, red, and far-red FPs are cytotoxic...
moreFluorescent proteins (FPs) are invaluable tools for biomedical research. Useful FPs have desirable fluorescence properties such as brightness and photostability, but a limitation is that many orange, red, and far-red FPs are cytotoxic when expressed in the cytosol. This cytotoxicity stems from aggregation. To reduce aggregation, we engineered the surface of DsRed-Express to generate DsRed-Express2, a highly soluble tetrameric FP that is noncytotoxic in bacterial and mammalian cells. Directed evolution of DsRed-Express2 yielded the color variants E2-Orange, E2-Red/Green, and E2-Crimson. These variants can be used to label whole cells for single- and multi-color experiments employing microscopy or flow cytometry. Methods are described for reducing the higher-order aggregation of oligomeric FPs and for analyzing FP cytotoxicity in Escherichia coli and HeLa cells.
Fluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells...
moreFluorescent proteins (FPs) are extremely useful tools for whole-cell, tissue, and animal labeling. For these purposes, FPs may be monomeric or oligomeric, but should meet the criteria of being tolerated at high expression levels in cells and having desirable photophysical properties. Our goal was to create a variant of DsRed-Express that maintains the brightness, fast-maturation, and photostability of this protein,
GFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without...
moreGFP and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro-evolved RNA mimic of GFP, which as genetically encoded fusions makes possible live-cell, real-time imaging of biological RNAs without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we solved the cocrystal structure of Spinach bound to its cognate exogenous chromophore, showing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex and an unpaired G. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure guided the design of a miniaturized 'Baby Spinach', and it provides a foundation for structure-driven design and tuning of fluorescent RNAs.
Genetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small...
moreGenetically encoded fluorescent sensors can be valuable tools for studying the abundance and flux of molecules in living cells. We recently developed a novel class of sensors composed of RNAs that can be used to detect diverse small molecules and untagged proteins. These sensors are based on Spinach, an RNA mimic of GFP, and they have successfully been used to image several metabolites and proteins in living bacteria. Here we discuss the generation and optimization of these Spinach-based sensors, which, unlike most currently available genetically encoded reporters, can be readily generated to any target of interest. We also provide a detailed protocol for imaging ADP dynamics in living Escherichia coli after a change from glucose-containing medium to other carbon sources. The entire procedure typically takes ∼4 d including bacteria transformation and image analysis. The majority of this protocol is applicable to sensing other metabolites and proteins in living bacteria.
... A noncytotoxic DsRed variant for whole-cell labeling. Author(s): Rita L. Strack; Daniel E. Strongin; Dibyendu Bhattacharyya; Wen Tao; Allison Berman; Hal E. Broxmeyer; Robert J. Keenan MD; Benjamin S. Glick. Quantitative ...
