Article ; Online: RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes.
2021 Volume 658, Page(s) 137–160
Abstract: The past decade has seen an exponential increase in the identification of individual nucleobases that undergo base conversion and/or modification in transcriptomes. While the enzymes that catalyze these types of changes have been identified, the global ... ...
| Abstract | The past decade has seen an exponential increase in the identification of individual nucleobases that undergo base conversion and/or modification in transcriptomes. While the enzymes that catalyze these types of changes have been identified, the global interactome of these modifiers is still largely unknown. Furthermore, in some instances, redundancy among a family of enzymes leads to an inability to pinpoint the protein responsible for modifying a given transcript merely from high-throughput sequencing data. This chapter focuses on a method for global identification of transcripts recognized by an RNA modification/editing enzyme via capture of the RNAs that are bound in vivo, a method referred as RNA immunoprecipitation (RIP). We provide a guide of the major issues to consider when designing a RIP experiment, a detailed experimental protocol as well as troubleshooting advice. The RIP protocol presented here can be readily applied to any organism or cell line of interest as well as both RNA modification enzymes and RNA-binding proteins (RBPs) that regulate RNA modification levels. As mentioned at the end of the protocol, the RIP assay can be coupled to high-throughput sequencing to globally identify bound targets. For more quantitative investigations, such as how binding of an RNA modification enzyme/regulator to a given target changes during development/in specific tissues or assessing how the presence or absence of RNA modification affects transcript recognition by a particular RBP (irrespective of a role for the RBP in modulating modification levels); the RIP assay should be coupled to quantitative real-time PCR (qRT-PCR). |
|---|---|
| MeSH term(s) | High-Throughput Nucleotide Sequencing ; Immunoprecipitation ; RNA/genetics ; RNA/metabolism ; RNA Editing ; RNA-Binding Proteins/genetics ; RNA-Binding Proteins/metabolism |
| Chemical Substances | RNA-Binding Proteins ; RNA (63231-63-0) |
| Language | English |
| Publishing date | 2021-07-14 |
| Publishing country | United States |
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. |
| ISSN | 1557-7988 |
| ISSN (online) | 1557-7988 |
| DOI | 10.1016/bs.mie.2021.06.005 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
More links
Kategorien
Order via subito
This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.