Biotinylated tags for recovery and characterization of ribonucleoprotein complexes.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Penalva, LOF; Keene, JD

Published Date

  • October 2004

Published In

Volume / Issue

  • 37 / 4

Start / End Page

  • 604 - 610

PubMed ID

  • 15517973

Pubmed Central ID

  • 15517973

Electronic International Standard Serial Number (EISSN)

  • 1940-9818

International Standard Serial Number (ISSN)

  • 0736-6205

Digital Object Identifier (DOI)

  • 10.2144/04374st05

Language

  • eng