Single-molecule pull-down for investigating protein–nucleic acid interactions

Abstract

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined singlemolecule fluorescence with various protein complex pull-down techniques and determined the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes. Highlights : Single-molecule pull-down provides a real-time view of the interactions between protein complexes and nucleic acids. Single-molecule pull-down can be used to determine the stoichiometry of nucleoprotein complexes. Tandem affinity purification yields protein complexes that are suitable for singlemolecule kinetics studies. Various tactics have to be considered for the surface immobilization of nucleoprotein complexes.

TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments

“…3g). These data are consistent with previous reports that TRBP increases the pre-miRNA-binding affinity of Dicer1220.…”

“…This required the purification of the Dicer-TRBP as a protein complex. We used SIMPlex (single-molecule approach to immunoprecipitated protein complexes), a technique that we previously developed19, but improved the purification and immobilization scheme by using biotin as a tag20. In vivo biotinylated Dicer complexes were pulled down using Flag-beads, and Dicer immunoprecipitates (IPs) were immobilized on a surface via biotin–NeutrAvidin conjugation (Fig.…”

SRSF3 recruits DROSHA to the basal junction of primary microRNAs

“…This process involves two consecutive cleavages by RNase III-type enzymes. The primary miRNA transcript (pri-miRNA) synthesized in the nucleus is initially cleaved by the Microprocessor complex composed of nuclear RNase III DROSHA and its cofactor DGCR8 (Denli et al 2004;Gregory et al 2004;Han et al 2004Han et al , 2006Landthaler et al 2004;Auyeung et al 2013;Nguyen et al 2015;Fareh et al 2016;Herbert et al 2016;Kwon et al 2016). The resulting product, precursor miRNA (pre-miRNA) is exported to the cytoplasm and is further cleaved by another RNase III enzyme, Dicer, that produces a miRNA duplex of ∼22 nt (Ma et al 2004;MacRae et al 2006a,b;Park et al 2011;Gu et al 2012;Tian et al 2014).…”

Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

“…In this study, our system can also be utilized to tag biotinylated target proteins with organic dyes using commercially available organic-dye labeled streptavidin. Moreover, in addition to studying DNA–protein interactions by tethering single- or double-strand DNA on a functionalized coverslip (Redding et al, 2015 ; Ticau et al, 2015 ; Yeeles et al, 2015 ; Qi and Greene, 2016 ), it is also an elegant strategy to study protein–protein or RNA–protein interactions by tethering biotinylated proteins on a coverslip through a biotin–streptavidin linkage (Lu et al, 2015a , b ; Fareh et al, 2016 ). Therefore, we are confident that with the rapid development and wide application of single-molecule microscopy, the method described in this paper will be of great benefit to future research in this field.…”

“…In recent years, the rapidly developing technique of single-molecule fluorescence microscopy has made great contributions to revealing the details of nucleic acid and protein interactions in various biological processes, such as DNA replication (Yardimci et al, 2012 ; Duzdevich et al, 2015 ; Ticau et al, 2015 ; Graham et al, 2017 ), spliceosome (Fareh et al, 2016 ), and CRISPR-Cas systems (Redding et al, 2015 ).…”