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A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.

ABSTRACT: Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we detailed the dynamics of the CGG trinucleotide repeat-containing 'toxic RNA' associated with Fragile X-associated tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.

SUBMITTER: Strack RL

PROVIDER: S-EPMC3852148 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.

Strack Rita L RL Disney Matthew D MD Jaffrey Samie R SR

Nature methods 20131027 12

Imaging RNA in living cells is a challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with Spinach, an 'RNA mimic of GFP'. We found that Spinach was dimmer than expected when used to tag constructs in living cells owing to a combination of thermal instability and a propensity for misfolding. Using systematic mutagenesis, we generated Spinach2 that overcomes these issues and can be used to image diverse RNAs. Using Spinach2, we ...[more]

PMID: 24162923

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Project description:GW182 (also known asTNRC6) family members are critically involved in the final effector phase of miRNA-mediated mRNA repression. The three mammalian paralogs, TNRC6a, b and c, are thought to be redundant based on Argonaute (Ago) binding, tethering assays, and RNAi silencing of individual members in cell lines. To test this idea, we generated TNRC6a, b and c knockout mice. TNRC6a mutants die at mid-gestation, while b- and c- deleted mice are born at a Mendelian ratio. However, the majority of TNRC6b and all TNRC6c mutants die within 24h after birth, the latter with respiratory failure. Necropsy of TNRC6c mutants revealed normal-appearing airways that give rise to abnormally thick-walled distal gas exchange sacs. Immunohistological analysis of mutant lungs demonstrated a normal distribution of bronchiolar and alveolar cells, indicating that loss of TNRC6c did not abrogate epithelial cell differentiation. The cellular kinetics and relative proportions of endothelial, epithelial, and mesenchymal cells were also not altered. However, the underlying capillary network was simplified and endothelial cells had failed to become tightly apposed to the surface epithelium in TNRC6c mutants, presumably causing the observed respiratory failure. TGFβ family mutant mice exhibit a similar lung phenotype of thick-walled air sacs and neonatal lethality, and qRT-PCR confirmed dynamic downregulation of TGFβ1 and TGFβR2 in TNRC6c mutant lungs during sacculation. VEGFR, but not VEGF-A ligand, was also lower, likely reflecting the overall reduced capillary density in TNRC6c mutants. Together, these results demonstrate that GW182 paralogs are not functionally redundant in vivo. Surprisingly, despite regulating a general cellular process, TNRC6c is selectively required only in the distal lung and not until late in gestation for proper expression of the TGFβ family genes that drive sacculation. These results imply a complex and indirect mode of regulation of sacculation by TNRC6c, mediated in part by dynamic transcriptional repression of an inhibitor of TGFβ family gene expression.

Dataset Information

A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.

Publications

A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA.

Similar Datasets

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