Unbiased dynamic characterization of RNA-protein interactions by OOPS

Abstract

27Current methods for the identification of RNA-protein interactions require a quantity and 28 quality of sample that hinders their application, especially for dynamic biological 29 systems or when sample material is limiting. Here, we present a new approach to enrich 30 RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is 31 compatible with downstream proteomics and RNA sequencing. OOPS enables recovery 32 of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in 33 an unbiased manner. By applying OOPS to human cell lines, we extract the majority of 34 known RBPs, and importantly identify additional novel RBPs, including those from 35 previously under-represented cellular compartments. The high yield and unbiased 36 nature of OOPS facilitates its application in both dynamic and inaccessible systems. 37 Thus, we have identified changes in RNA-protein interactions in mammalian cells 38 following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. 39 Overall, OOPS provides an easy-to-use and flexible technique that opens new 40 opportunities to characterize RNA-protein interactions and explore their dynamic 41 behaviour. 42 43 44 59 conditions 13 . Moreover, the theoretical dependence on the presence of RNA polyA tails, 60 makes oligo(dT)-based capture methods incompatible for organisms with little or no 61 polyadenylation, such as prokaryotes, or for non-polyadenylated RNAs; thus introducing 62 bias in the RBPs identified. There have been recent attempts to address the oligo(dT) 63 limitations, but these have involved incorporation of modified nucleotides, which by itself 64 restricts its applicability and introduces a bias in transcription-dependent nucleoside-65 incorporation 14-16 . 66 67 2 To address these limitations, we have developed a method based on Acidic 68 Guanidinium Thiocyanate-Phenol-Chloroform (AGPC) phase partition, called 69 Orthogonal Organic Phase Separation (OOPS). AGPC purification permits the recovery 70 of RNA species in an unbiased manner 17,18 . In standard conditions, when lysing cells in 71 AGPC, two distinct phases are formed: RNA migrates to the upper aqueous phase and 72 proteins occupy the lower organic phase. Here, we utilise UV crosslinking at 254 nm to 73 generate RNA-protein adducts that combine the physicochemical properties of both 74 molecules and thus migrate to the aqueous-organic interface 19 . Isolation of the interface 75 allows specific recovery of either RBPs or PBRs by digesting the reciprocal component 76 of the adduct. 77 78 Here, we demonstrate the specificity and versatility of OOPS. Separation of free and 79 protein-bound RNA provides a way to quantify the proportion of RNA crosslinked to 80 protein, enabling precise optimisation of UV dosage. Furthermore, we show that OOPS 81 recovers all crosslinked-RNA (CL-RNA) and thus all crosslinked RBPs. To demonstrate 82 the versatility of OOPS, we analyse RNA-binding changes through cell-cycle 83 progression following noc...

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

“…The identification of five new RNP condensates associated with BR-bodies suggests these structures likely have a broader role in bacterial cell organization than previously anticipated. Indeed, recent high-throughput methods designed to detect proteins crosslinked to RNA identified >1100 RNA binding proteins in E. coli including many metabolic enzymes 55,56 . Indeed, we identified two metabolic enzymes, MetK and FabG, which have the capacity to phase separate with RNA (Fig 3).…”

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…These steps were repeated and peptides were dried in a speed vac. Samples for glycopeptide enrichment were digested in-solution according to (Queiroz et al, 2019). Samples were reduced and alkylated in 10 mM DTT and 50 mM iodoacetamide.…”

“…Samples were dried and finally, peptides were resuspended in 20 μl 0.1 % FA. For glycopeptide enrichment peptides were first desalted using poros oligo r3 resin (1-339-09, Thermo Scientific TM , Bishop's Stortford, United Kingdom) as described (Gobom et al, 1999;Queiroz et al, 2019). Pierce™ centrifuge columns (SH253723, Thermo Scientific TM , Bishop's Stortford, United Kingdom) were filed with 250 μl of poros oligo r3 resin.…”

Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World

“…Very recently three similar strategies, named respectively OOPS, XRNAX, and PTex, have been developed as very promising global approaches to expand the current knowledge on RBPs and RNA-binding domains (Queiroz et al, 2018 ; Trendel et al, 2018 ; Urdaneta et al, 2018 ). They all adopt an organic phase separation step for the extraction and enrichment of UV-crosslinked RNA-protein complexes from whole cell extracts.…”