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

Abstract

As part of their normal life cycle, most RNA molecules associate with several proteins that direct their fate and regulate their function. Here, we describe a novel method for identifying proteins that associate with a target RNA. The procedure is based on the ARiBo method for affinity purification of RNA, which was originally developed to quickly purify RNA with high yields and purity under native conditions. The ARiBo method was further optimized using in vitro transcribed RNA to capture RNAassociating proteins from cellular extracts with high yields and low background protein contamination. For these RNA pulldowns, stem-loops present in the immature forms of let-7 miRNAs (miRNA stem-loops) were used as the target RNAs. Labelfree quantitative mass spectrometry analysis allowed for the reliable identification of proteins that are specific to the stemloops present in the immature forms of two miRNAs, let-7a-1 and let-7g. Several proteins known to bind immature forms of these let-7 miRNAs were identified, but with an improved coverage compared to previous studies. In addition, several novel proteins were identified that better define the protein interactome of the let-7 miRNA stem-loops and further link let-7 biogenesis to important biological processes such as development and tumorigenesis. Thus, combining the ARiBo pull-down method with label-free quantitative mass spectrometry provides an effective proteomic approach for identification of proteins that associate with a target RNA.

Discovery of a Well-Folded Protein Interaction Hub Within the Human Long Non-Coding RNANORAD

“…Therefore, it is important to purify NORAD RNA domains under non-denaturing conditions for any biophysical or chemical studies. To identify the protein binding partners of specific domains in NORAD, we devised a strategy consisting of in vitro RNA-pull downs coupled with label-free quantitative mass spectrometry, using purified RNA under native conditions (Ilagan and Jurica 2014, Di Tomasso, Miller Jenkins et al 2016) (Figure 3A).…”

RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

“…A second class of aptamers includes short RNA hairpins that interact specifically with proteins, such as the coat proteins from the R17/MS2 bacteriophage [ 49 ], bacterial streptavidin S1 [ 50 ], the PP7 coat protein [ 51 ], the lambda bacteriophage anti-terminator protein N (or lambdaN peptide) [ 52 ], an engineered version of the Csy4 endonuclease [ 53 ], or artificial pentatricopeptide repeat (PPR) proteins [ 54 ]. While this class of RNA aptamers has been broadly used to globally determine proteins/RNAs interacting with the tagged RNAs in vitro (e.g., [ 52 , 55 ]; reviewed in [ 36 , 42 , 56 ]); the RNA aptamers and interacting proteins have also been coexpressed in vivo to recover endogenously formed RNP complexes from cell lysates through affinity capture [ 49 , 57 , 58 , 59 ]. For example, RBP purification and identification (RaPID) implemented the affinity purification of MS2 aptamer-tagged RNAs and detection of bound proteins and transcripts with MS and reverse transcription (RT)-PCR, respectively [ 49 ].…”

Drosophila mRNA Localization During Later Development: Past, Present, and Future

“…Many of the proteins known to localize during oocyte or early embryo development are expressed in one or more lineages in later development. Notably, there have been several advances in identifying protein binding to specific RNAs that would be compatible for use in complex tissues (Lee et al, 2013 ; Di Tomasso et al, 2016 ; Autour et al, 2018 ), reviewed in (Faoro and Ataide, 2014 ).…”