Specific RNP capture with antisense LNA/DNA mixmers

Abstract

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

Approaches for Understanding the Mechanisms of Long Noncoding RNA Regulation of Gene Expression

“…The other general approach for RNA purification is based on hybridization of antisense oligonucleotides (Chu et al 2011;Engreitz et al 2013;Simon et al 2013;Rogell et al 2017). These approaches generally use biotin labeled oligonucleotides to hybridize to a specific RNA sequence and capture it, and its associated proteins, from a complex mixture.…”

“…These approaches generally use biotin labeled oligonucleotides to hybridize to a specific RNA sequence and capture it, and its associated proteins, from a complex mixture. These strategies vary in several ways: (1) some use individual oligonucleotides (Simon et al 2013;Rogell et al 2017) whereas others use pools of oligonucleotides that tile across the RNA of interest (Chu et al 2011;Engreitz et al 2013); (2) some methods use short oligonucleotides (Chu et al 2011;Simon et al 2013;Rogell et al 2017) and others use long oligonucleotides (Engreitz et al 2013); and (3) some use unmodified nucleotides (Chu et al 2011;Engreitz et al 2013;Simon et al 2013), whereas others use locked nucleic acids (Rogell et al 2017). The key consideration in choosing between these parameters comes down to specificity of the hybrid and the stringency of the purification conditions used in the purification procedure.…”

Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

“…10,11 Antisense oligonucleotides have also been demonstrated using a two-step purification of mRNA transcripts with a preliminary polyA purification, in addition to the analysis of ribosomal RNA. 12,13 While these approaches extend the utility of antisense oligonucleotides, ribosomal RNA is extremely abundant, and the two-step approach relies on polyA purification, and thus is not applicable to nonpolyadenylated RNAs. Furthermore, optimization of ASO sequence and consideration of RNA base pairing availability is required.…”

β-actin mRNA interactome mapping by proximity biotinylation

“…However, the major approaches to determine which proteins associate with a specific RNA are affinity purification of modified or tagged RNAs together with their bound proteins, or co-immunoprecipitation of RNP components with the help of known RBPs (28). In addition, affinity capturing of specific RNPs with hybridizing antisense probes or via integrated aptamers has been successfully used (29)(30)(31). A limitation of these techniques is the potential loss of low affinity binders during purification, which has so far been addressed by in vivo UV cross-linking prior to cell lysis (25,26).…”