Single‐molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation

Abstract

Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…New single-molecule approaches [14][15][16][17] will enable biologists to explore protein complexes at the nanoscale and will provide opportunities for tackling challenges that require more physiologically relevant systems than purified proteins.…”

“…Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] …”

“…However, these approaches are static and cannot provide reliable stoichiometric or dynamic information. To deal with this limitation, two teams have developed a promising approach based on singlemolecule IP [16,17]. In this approach, streptavidin is layered via biotin-streptavidin binding on a polymer-coated glass surface, and biotinylated antibodies are conjugated to the streptavidin layer (Figure 4a).…”

“…Using cell extracts, Gelles and colleagues visualized spliceosome assembly [14,19], and van Oijen and colleagues reconstituted a eukaryotic replisome on a single-molecule surface [15,20]. Using protein immunoprecipitates, Joo and colleagues have studied the regulation process of microRNA biogenesis [16], and Ha and colleagues have demonstrated single-molecule complex analysis [17,21]. These new approaches have a key advantage over conventional in vitro single-molecule techniques: the proteins continuously interact with other cellular proteins, spontaneously form a macromolecular complex, and dissociate during observation.…”

“…They then introduced a microRNA substrate labeled with a dye and bound by its cofactor (Lin28) onto this surface. The uridylation of the microRNA substrate was visualized in real time [16]. By recording weak interactions between the polymerase complex and the RNA substrate, which could not have been detected from a bulk measurement, they discovered that the cofactor protein Lin28 is a processivity factor that enhances the binding affinity between the poly(U) polymerase and its microRNA substrate.…”