RNAs and ribonucleoprotein complexes (RNPs) play central roles in biology and disease. Despite their importance to both basic and clinical biology, there is no general methodology to study the composition and function of RNPs isolated from their native context. To address this need, our Phase I goal is to develop a new and robust affinity separation technology for RNP complexes. In Phase II, we propose to develop the method into an easy-to-use kit that can be employed by researchers to detect and analyze specific RNPs in their laboratories without special expertise for a variety of applications. Our strategy is based on an endoribonuclease that binds a specific RNA sequence with very high affinity and cleaves this sequence at a single site. A mutant version of this protein can bind but not cleave target RNA. In our method, a target RNA will be expressed in vitro or in vivo with a 5' tag composed of the endoribonuclease target sequence. Cellular extracts will be applied to the mutant endoribonuclease immobilized on resin. The tagged RNA will be trapped by the mutant endoribonuclease. Under specific buffer conditions, cleavage activity of the mutant endoribonuclease can be reactivated, causing the tag to be cleaved off, liberating the target RNA and any protein partners bound to it. The isolated RNA and its associated RNA binding proteins can then be analyzed by a variety of molecular biological or biophysical techniques.
Public Health Relevance Statement: Public Health Relevance: Recent deep sequencing experiments have revealed that as much as 20 times more of the human genome is transcribed into noncoding RNAs (RNAs that don't get translated into proteins) than is transcribed into protein-coding messenger RNAs. Many of these noncoding RNAs have been implicated in various cancers and diseases, but little is known about their normal or disease-related cellular functions because there is no general method for purifying and studying these RNAs and the proteins to which they bind. By developing an RNA affinity purification technology, we can begin to shed light on the normal functions of these RNAs and how they may play a role in various diseases.
Project Terms: Address; Affinity; Affinity Chromatography; Avidin; base; Base Sequence; Binding (Molecular Function); Biological; Biological Assay; Biology; Biotin; Buffers; Cell Extracts; Cell physiology; Cells; Cleaved cell; Clinical; Code; Complex; deep sequencing; Disease; Elements; endoribonuclease; Endoribonucleases; Engineering; Ensure; Genetic Transcription; Goals; Hour; Human Genome; Imidazole; Immune system; In Vitro; in vivo; Incubated; Laboratories; Length; Light; Little's Disease; Malignant Neoplasms; Messenger RNA; Methodology; Methods; Molecular; Molecular Biology; mutant; Nucleotides; Phase; Plant Resins; Plasmids; Play; Proteins; public health relevance; Reaction; Research Personnel; research study; Ribonucleoproteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Role; Sampling; Site; Solid; Solutions; Specificity; Structure; Techniques; Technology; Temperature; Testing; Time; Transcript; Transfection; Translating; Untranslated RNA
