Large-Scale Immunopurification of Ribonucleoprotein Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays

Abstract

It is of interest to be able to define sets of cellular RNAs associated with specific RNA-binding proteins. This "guilt by association" can lead to new insights into how RNA-binding proteins modulate posttranscriptional gene expression of specific target RNAs. To identify these RNAs, antibodies against RNA-binding proteins can be used to immunopurify endogenous RNA-protein complexes from cells, and then the associated RNAs can be characterized. The method described here was developed to identify binding regions on nuclear transcripts for Drosophila heterogeneous nuclear ribonucleoproteins (hnRNPs). An antibody is added to an RNP extract and incubated to allow antigen-antibody complexes to form. The antibody-antigen complexes are then retrieved by binding of the antibody constant region to staphylococcal Protein A immobilized on Sepharose beads. The bead-immobilized complexes are then washed and RNA is prepared. The RNA is used to generate random-primed cDNA, cRNA, and biotinlyated cDNA probes for use on Affymetrix whole-genome Drosophila tiling arrays.

Preparation of Ribonucleoprotein-Enriched Nucleoplasmic Extracts from Drosophila Cells

“…The associated RNA can be isolated and used for microarray analysis (also known as RIP-Chip) (Labourier et al 2002;Blanchette et al 2004Blanchette et al , 2009Olson et al 2007) or high-throughput RNA sequencing (RIP-Seq). For a sample application, see Large-Scale Immunopurification of Ribonucleoprotein Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays (Rio 2014).…”

Analysis of RNA Metabolism in Fission Yeast

“…RIP-chip refers to a strategy in which RNAs in the immunoprecipitate are identified by microarray analysis. The procedure for fission yeast (Amorim et al 2010;Duncan and Mata 2011;Hasan et al 2014) is similar overall to the one used to characterize nuclear ribonucleoprotein particles from Drosophila (see Protocol: Large-Scale Immunopurification of Ribonucleoprotein Complexes from Drosophila Nucleoplasmic Extracts for Tiling Microarrays [Rio 2014c]), mainly differing in the use of whole cell rather than fractionated lysates. High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) or its variations are now commonly used in other organisms (Darnell 2010), and a CRAC (cross-linking and analysis of cDNA) protocol (similar to HITS-CLIP) used in budding yeast has been adapted by Kilchert et al (2015) to investigate fission yeast RNAs bound to the Mmi1 protein, which targets them for destruction by the exosome.…”