A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

• Tandem RNA isolation procedure (TRIP) to enrich particular mRNAs and bound proteins.
• TRIP uses modified antisense RNA oligonucleotides annealing with sequences in mRNA.
• Adaptable to native cells/organism without requirement for genetic engineering.

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3′-biotinylated 2′-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues.