Biotinylated Tags for Recovery and Characterization of Ribonucleoprotein Complexes
Year: 2004
Type: article
Abstract: Determining the in vivo targets of RNA-binding proteins and characterizing the posttranscriptional networks in which they participate constitute major challenges in the post-genomic era. An important step in this direction is the development of methods that permit efficient recovery of ribonucleoprotein (RNP) complexes. We present an improved methodology for efficient isolation of mammalian cell RNPs in which a biotin acceptor peptide (BAP) is used to tag RNA-binding proteins. BAP-tagged RNA-binding proteins can be biotinylated in vivo by co-expression of the Escherichia coli BirA enzyme. RNP recovery was obtained using streptavidin sepharose beads, and messenger RNAs (mRNAs) were identified using multiprobe RNase protection assays and cDNA microarrays. Using this approach we efficiently recovered and quantified RNAs bound to cytoplasmic poly(A)-binding protein (PABP) and to nuclear human transformer 2 (hTra-2) with minimal background.
Source: BioTechniques
DOI: 10.2144/04374st05
Authors: Luiz O.F. Penalva, Jack D. Keene
Institution: Duke Medical Center
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
OpenAlex ID: W1913111655