In vivo expression and purification of aptamer-tagged small RNA regulators

Abstract

Small non-coding RNAs (sRNAs) are an emerging class of post-transcriptional regulators of bacterial gene expression. To study sRNAs and their potential protein interaction partners, it is desirable to purify sRNAs from cells in their native form. Here, we used RNA-based affinity chromatography to purify sRNAs following their expression as aptamer-tagged variants in vivo. To this end, we developed a family of plasmids to express sRNAs with any of three widely used aptamer sequences (MS2, boxB, eIF4A), and systematically tested how the aptamer tagging impacted on intracellular accumulation and target regulation of the Salmonella GcvB, InvR or RybB sRNAs. In addition, we successfully tagged the chromosomal rybB gene with MS2 to observe that RybB-MS2 is fully functional as an envelope stress-induced repressor of ompN mRNA following induction of sigmaE. We further demonstrate that the common sRNA-binding protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. The presented affinity purification strategy may facilitate the isolation of in vivo assembled sRNA–protein complexes in a wide range of bacteria.

Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq

“…S8A for a schematic view of the MS2-tagged construct). This approach has been used successfully to demonstrate Hfq binding on MS2-tagged sRNAs (Said et al 2009). Each construct was cloned downstream from an arabinose-inducible promoter carried on a low-copy-number plasmid (see the Materials and Methods for details).…”

“…Affinity purification of MS2-tagged RNA Affinity purification assays were performed as described (Said et al 2009), with some modifications. The MS2-MBP protein was purified as described in the Supplemental Material.…”

ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry

“…However, there is also a need to characterize RNA-protein networks from the vantage point of the RNA, and a few strategies have been developed to purify RNA-protein complexes using affinity-tagged RNAs. Although most of these AP-MS investigations using tagged RNAs are based on in vitro assembly with proteins from cellular extracts, the characterization of RNA-protein complexes formed in vivo has also been investigated (Hogg and Collins 2007a,b;Vasudevan and Steitz 2007;Said et al 2009;Baltz et al 2012;Castello et al 2012;Oeffinger 2012;Kwon et al 2013;Faoro and Ataide 2014;Gerstberger et al 2014;Kotzer-Nevo et al 2014;Leppek and Stoecklin 2014;McHugh et al 2014;Beckmann et al 2015;Dong et al 2015). Despite these efforts, there is still much that remains to be learned about the protein-interaction networks of specific RNAs.…”

A game of tag: MAPS catches up on RNA interactomes

“…15,16 Later, the Vogel group developed an in vivo method to pull-down specific sRNAs and identify their protein partners. 17,18 After production of a MS2-tagged sRNA, cell lysates are applied to an amylose resin on which a MBP-MS2 protein (Maltose Binding Protein fused with the MS2 coat protein) has been immobilized. Following elution by addition of a competitor for binding to the MBP, samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS).…”

“…This technique was revolutionary as it was performed in vivo and didn't require addition of external proteins or molecules to achieve adequate purification. 17,18 Notably, this method allowed purification and identification of a protein found in most sRNA-protein complexes, the RNA chaperone Hfq. The protein Hfq is known to interact with sRNAs to stabilize them in vivo and to facilitate sRNA-mRNA complexes formation.…”