Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity. Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. A widespread self-cleaving ribozyme class is revealed by bioinformatics. Metazoan tRNA introns generate stable circular RNAs in vivo. Broccoli: Rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. Archaeal 3′-phosphate RNA splicing ligase characterization identifies the missing component in tRNA maturation. RtcB is the RNA ligase component of an Escherichia coli RNA repair operon. HSPC117 is the essential subunit of a human tRNA splicing ligase complex. Novel mechanism of RNA repair by RtcB via sequential 2′,3′- cyclic phosphodiesterase and 3′-phosphate/5′-hydroxyl ligation reactions. Origin of splice junction phosphate in tRNAs processed by HeLa cell extract.
Characterization of tRNA precursor splicing in mammalian extracts. Ribonuclease ‘XlaI,’ an activity from Xenopus laevis oocytes that excises intervening sequences from yeast transfer ribonucleic acid precursors. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. Circular RNAs are abundant, conserved, and associated with ALU repeats. CircRNA biogenesis competes with pre-mRNA splicing. Developing fluorogenic riboswitches for imaging metabolite concentration dynamics in bacterial cells. Imaging metabolite dynamics in living cells using a Spinach-based riboswitch. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. Fluorescence imaging of cellular metabolites with RNA. Genetically encoded sensors to elucidate spatial distribution of cellular zinc. In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies.
Expression of small, therapeutic RNAs in human cell nuclei.
Conformationally selective RNA aptamers allosterically modulate the beta2-adrenoceptor. RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of β-secretase BACE1 in vitro. Rentmeister, A., Bill, A., Wahle, T., Walter, J. In vitro selection of RNA molecules that bind specific ligands. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. The Tornado expression system thus markedly enhances the utility of RNA-based approaches in the mammalian cell. Additionally, an RNA-based fluorescent metabolite biosensor for S-adenosyl methionine (SAM) that is expressed at low levels when expressed as a linear RNA achieves levels sufficient for detection of intracellular SAM dynamics when expressed as a circular RNA. Using this approach, protein-binding aptamers that otherwise have minimal effects in cells become potent inhibitors of cellular signaling. The ribozymes rapidly undergo autocatalytic cleavage, leaving termini that are ligated by the ubiquitous endogenous RNA ligase RtcB. Tornado-expressed transcripts contain an RNA of interest flanked by Twister ribozymes. Here we describe the Tornado (Twister-optimized RNA for durable overexpression) expression system for achieving rapid RNA circularization, resulting in RNA aptamers with high stability and expression levels. However, their usefulness in the mammalian cell is limited by low expression and rapid degradation. RNA aptamers and RNA aptamer-based devices can be genetically encoded and expressed in cells to probe and manipulate cellular function.
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