Ribonucleoprotein purification and characterization using RNA Mango

Abstract

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

Current approaches for RNA-labelling to identify RNA-binding proteins

“…The RNA Mango aptamer (Fig. 3H) was designed to provide another option for affinity-based native purification methods of associated RNPs with the added benefit of fluorescence detection (Panchapakesan et al 2017). This RNA aptamer consists of a 19 nt fluorophore-binding domain that has a high affinity (K d 3.2 nmol/L) to fluorophore reagents: thiazole orange (TO), TO1-biotin, or TO1-desthiobiotin (Trachman et al 2017;Xia et al 2017).…”

“…This RNA aptamer consists of a 19 nt fluorophore-binding domain that has a high affinity (K d 3.2 nmol/L) to fluorophore reagents: thiazole orange (TO), TO1-biotin, or TO1-desthiobiotin (Trachman et al 2017;Xia et al 2017). The RNA of interest is synthesized by in-vitro transcription with the Mango aptamer tag, and is then incubated in the cellular extract to allow RNP complexes to form (Panchapakesan et al 2017). The extract containing the Mango-tagged RNA is incubated in streptavidin solid support resin conjugated to TO1-desthiobiotin.…”

“…The RNP complex can be visualized using SDS-PAGE analysis, further purified using size-exclusion chromatography, and RNA-binding protein identity can be confirmed using liquid chromatography -mass spectrophotometry (LC-MS). Downstream characterization studies between the RNA and RBP can also utilize the fluorescent tag by using single-molecule fluorescence cross-correlation spectroscopy, or other methods (Panchapakesan et al 2017).…”

RNA2DMut: a web tool for the design and analysis of RNA structure mutations

“…Y RNA localization and interactions are of great interest; therefore the example task performed on hY5 was to identify the optimal site for introducing an MS2 aptamer sequence/structure. RNA aptamers are useful tools for both purification and characterization (e.g., imaging) of RNAs (Panchapakesan et al 2017). For example, MS2 aptamers were previously introduced into the EBER1 ncRNA (Lee et al 2012) to facilitate capture of endogenously interacting host proteins (via binding of the MS2 aptamer by MS2 bacteriophage coat protein [Stockley et al 1995]).…”

Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes

“…RNA-based affinity purification systems have been valuable tools for the isolation and characterization of endogenously assembled RNPs (Slobodin and Gerst 2010;Yoon and Gorospe 2016;Panchapakesan et al 2017). In particular, a system based on the Pseudomonas phage 7 (PP7) coat protein and its cognate hairpin binding site has been used to investigate proteins associated with noncoding RNAs and the targeted degradation of transcripts by the nonsense-mediated mRNA decay pathway (Hogg and Collins 2007a,b;Hogg and Goff 2010;Ge et al 2016).…”