RNA Affinity Tags for the Rapid Purification and Investigation of RNAs and RNA–Protein Complexes

Abstract

SummaryIsolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using affinity purification methods that are specific for the RNA rather than the protein components of these complexes. We describe the use of two such RNA affinity tags: small RNAs that bind with high affinity and specificity to either Sephadex beads or streptavidin affinity resins and can be eluted under mild, native conditions that retain intact complexes. The tags can be inserted into appropriate locations in genes encoding the RNA components, and ribonucleoproteins can be assembled either in vivo or in vitro before affinity isolation. Strategies toward the design and production of these tagged RNA sequences are discussed, and the purification procedure is outlined.

mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry

“…3B, bar graph), indicating that rapamycin selectively disrupts the IMP2-L3 association. To examine this in a reciprocal manner, we appended to the L3-luciferase and L4-luciferase reporters immediately beyond the termination triplet an aptamer (S1) conferring the ability to bind streptavidin (Walker et al 2008) and expressed each stably in RD cells (Fig. 3C).…”

“…The IGFII L3 and L4 were inserted into the Hind III site upstream of the firefly luciferase coding region, which was followed by the S1 streptavidin-binding RNA motif (59-ACCGACCAGAAUCAUG CAAGUGCGUAAGAUAGUCGCGGGCCGGG-39) (Walker et al 2008) in pcDNA5/TO vector. The human RD cell lines stably expressing S1-L3 and S1-L4 growing up to 25%-30% confluence were cross-linked in 0.3% formaldehyde in PBS for 10 min at 37°C (Niranjanakumari et al 2002).…”

Translational regulation of the cell cycle: when, where, how and why?

“…RNA-affinity chromatography could lead to identification of additional RNA-binding proteins interacting with 5 0 UTRs or 3 0 UTRs of mRNAs [120,121]. This approach may elucidate some novel translational controls required for efficient translation of mRNAs encoding cell cycle regulators, which, as discussed above, in some cases require specialized translational initiation factors owing to their long and complex UTRs.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…In addition to reverse-phase HPLC [128], the use of affinity chromatography has been described [129][130][131][132]. Batey and Kieft developed a sophisticated approach, where an affinity tag is attached to the 3'-end of the RNA by a glucosamine-6-phosphate activated (glmS) ribozyme [130].…”

“…Elution of the RNA can be achieved by activating the ribozyme with addition of GlcN6P. Affinity purification based on aptamer tags binding Sephadex or Streptavidin have also been proposed [122,131].…”