Use of Aptamer Tagging to Identify In Vivo Protein Binding Partners of Small Regulatory RNAs
Small regulatory RNAs (sRNAs) are short, generally noncoding RNAs that act posttranscriptionally to control target gene expression. Over the past 10 years there has been a rapid expansion in the discovery and characterization of sRNAs in a diverse range of bacteria. Paradigm shifts in our understanding of the breadth of posttranscriptional control by sRNAs were achieved in a number of pioneering studies that involved immunoprecipitation of a known RNA chaperone, the near-ubiquitous Hfq, followed by sequencing to identify novel putative regulators and targets. To perform the converse experiment, we previously developed a method which uses an aptamer-tagged sRNA to allow purification of in vivo assembled RNA-protein complexes and subsequent identification of bound proteins. We successfully implemented this protocol using the Hfq-associated sRNA, InvR, tagged with a tandem repeat of the commonly used MS2-aptamer. Incorporation of the aptamer had no effect on sRNA stability or activity. InvR-MS2 could be effectively purified along with associated proteins, such as Hfq, using maltose binding protein fused to the MS2 coat protein (MBP-MS2) immobilized on an amylose column. Mass-spectroscopy was also used to identify previously uncharacterized protein partners. These results have been described previously (Said et al., Nucleic Acids Res 37:e133, 2009) and thus the figures presented here are intended solely as an illustrative guide to complement this detailed step-by-step protocol.
Available from: David Lalaouna
- "We fused the 5'-end of ITS metZ-metW and ITS metW-metV with bacteriophage MS2 RNA stemloops, which are bound by the MS2 coat protein with high specificity. This highly specific interaction was previously shown to allow affinity co-purification of sRNA-bound proteins from bacterial extracts (Corcoran et al., 2012). As controls, we used untagged ITS metZ-metW and ITS metW-metV expressed under the same conditions. "
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ABSTRACT: RNA sequencing (RNAseq) technology recently allowed the identification of thousands of small RNAs (sRNAs) within the prokaryotic kingdom. However, drawing the comprehensive interaction map of a sRNA remains a challenging task. To address this problem, we recently developed a method called MAPS (MS2 affinity purification coupled with RNA sequencing) to characterize the full targetome of specific sRNAs. This method enabled the identification of target RNAs interacting with sRNAs, regardless of the type of regulation (positive or negative), type of targets (mRNA, tRNA, sRNA) or their abundance. We also demonstrated that we can use this technology to perform a reverse MAPS experiment, where an RNA fragment of interest is used as bait to identify interacting sRNAs. Here, we demonstrated that RybB and MicF sRNAs co-purified with internal transcribed spacers (ITS) of metZ-metW-metV tRNA transcript, confirming results obtained with MS2-RybB MAPS.
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ABSTRACT: sRNAs (small non-coding RNAs) representing important players in many cellular and regulatory processes have been identified in all three domains of life. In Eukarya and Bacteria, functions have been assigned for many sRNAs, whereas the sRNA populations in Archaea are considerably less well characterized. Recent analyses on a genome-wide scale particularly using high-throughput sequencing techniques demonstrated the presence of high numbers of sRNA candidates in several archaea. However, elucidation of the molecular mechanism of sRNA action, as well as understanding their physiological roles, is in general still challenging, particularly in Archaea, since efficient genetic tools are missing. The identification of cellular targets of identified archaeal sRNAs by experimental approaches or computational prediction programs has begun only recently. At present, targets have been identified for one archaeal sRNA, sRNA162 in Methanosarcina mazei, which interacts with the 5' region of its targets, a cis-encoded and a trans-encoded target, blurring the paradigm of a border between cis- and trans-encoded sRNAs. Besides, the first experimental implications have been obtained in Haloarchaea and Pyrobaculum that archaeal sRNAs also target 3' regions of mRNAs. The present review summarizes our current knowledge on archaeal sRNAs and their biological functions and targets.
Available from: Abdullah Ozer
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ABSTRACT: Single-stranded oligonucleotide aptamers have attracted great attention in the past decade because of their diagnostic and therapeutic potential. These versatile, high affinity and specificity reagents are selected by an iterative in vitro process called SELEX, Systematic Evolution of Ligands by Exponential Enrichment. Numerous SELEX methods have been developed for aptamer selections; some that are simple and straightforward, and some that are specialized and complicated. The method of SELEX is crucial for selection of an aptamer with desired properties; however, success also depends on the starting aptamer library, the target molecule, aptamer enrichment monitoring assays, and finally, the analysis and characterization of selected aptamers. Here, we summarize key recent developments in aptamer selection methods, as well as other aspects of aptamer selection that have significant impact on the outcome. We discuss potential pitfalls and limitations in the selection process with an eye to aid researchers in the choice of a proper SELEX strategy, and we highlight areas where further developments and improvements are desired. We believe carefully designed multiplexed selection methods, when complemented with high-throughput downstream analysis and characterization assays, will yield numerous high-affinity aptamers to protein and small molecule targets, and thereby generate a vast array of reagents for probing basic biological mechanisms and implementing new diagnostic and therapeutic applications in the near future.
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