RNA-protein interactions play a key role in gene regulation, affecting processes from splicing and stability to translation. Despite their importance, traditional approaches for studying RNA-binding proteins (RBPs) have significant limitations. Many focus only on polyadenylated RNAs, leaving large classes of non-coding RNAs and their interactors untouched.
A team of researchers addressed this challenge by developing Orthogonal Organic Phase Separation (OOPS), a simple but powerful method that captures the entirety of cellular RNA-protein complexes, without relying on prior knowledge of RNA types or sequence features.
Study design: developing a universal capture method
The researchers adapted a classical biochemical principle: during acid guanidinium thiocyanate–phenol–chloroform (AGPC) extraction, proteins partition into the organic phase and RNA into the aqueous phase. When proteins and RNAs are covalently crosslinked, however, they migrate together to the interphase.
Building on this, the team created OOPS, which uses UV crosslinking followed by multiple rounds of AGPC separation to isolate pure RNA-protein complexes. After separation, the complexes can be treated with RNase to release proteins for mass spectrometry analysis, or with protease to release RNA for sequencing. This makes OOPS a versatile platform for both proteomics and transcriptomics.
Key findings
Using OOPS in human (HeLa) cells and mouse tissues, the researchers identified thousands of RNA-binding proteins, including many not previously annotated as RBPs. Importantly, the method captured interactions involving both coding and non-coding RNAs, extending well beyond the reach of poly(A)-dependent approaches such as oligo(dT) pulldown.
Among the discoveries were unexpected interactions between RNA and proteins traditionally considered metabolic enzymes, transcription factors, or structural components. These findings highlight the pervasive and multifunctional nature of RNA-protein interactions across the proteome.
By comparing OOPS with existing techniques like interactome capture, the study demonstrated that OOPS dramatically expands the detectable landscape of RNA-binding proteins.
Ensuring robust sequencing with effective rRNA depletion
A critical technical step in the study was the preparation of RNA for sequencing. Ribosomal RNA (rRNA) normally accounts for more than 80% of total RNA in cells, and if not removed efficiently, it can dominate sequencing libraries and obscure meaningful results. To overcome this, the researchers used Lexogen’s RiboCop kit, a highly effective solution for rRNA depletion.
By depleting rRNA, the team ensured that sequencing reads were enriched for messenger RNAs and non-coding RNAs of interest, rather than wasted on abundant ribosomal sequences. This step was particularly important in the context of OOPS, where capturing the full range of coding and non-coding RNAs was central to the study’s aims. High-quality rRNA depletion improved sensitivity; allowed rare RNA species to be detected more reliably; and ultimately strengthened the biological insights gained from the dataset.
Ensuring reproducibility and broad applicability
OOPS is not only comprehensive but also robust and widely accessible. Because it uses standard reagents and simple organic extraction steps, the protocol can be implemented in most molecular biology labs without specialised equipment. This makes it an attractive choice for large-scale projects, cross-species comparisons, and the discovery of novel RBPs in previously understudied systems.
The study also highlighted that applying OOPS across multiple tissues and cell types provided highly reproducible results, underscoring its value for generating reliable datasets in RNA biology.
Limitations and considerations
As with all UV crosslinking-based methods, efficiency depends on nucleotide–amino acid proximity and does not capture every RNA-protein interaction equally. Additionally, while OOPS identifies the proteins and RNAs involved, further steps are required to determine the precise binding sites or functional consequences of these interactions.
Nonetheless, the approach overcomes a key bottleneck in the field: the bias of traditional methods toward polyadenylated RNAs. By enabling unbiased capture, OOPS opens the door to systematic exploration of the full RNA-protein interactome.
Conclusions: a new standard for RNA interactome studies
The development of OOPS represents a significant advancement for RNA biology. By allowing researchers to isolate RNA-protein complexes comprehensively, reproducibly, and without bias toward RNA class, it provides a more complete picture of how RNAs and proteins interact to regulate cellular function.
The combination of innovative methods like OOPS with robust sequencing preparation tools such as RiboCop shows how careful experimental design enables scientists to tackle long-standing questions in molecular biology. Together, these approaches are helping uncover new layers of regulation across coding and non-coding transcriptomes.
References
Queiroz RML, Smith T, Villanueva E, Marti-Solano M, Monti M, Pizzinga M, Mirea DM, Ramakrishna M, Harvey RF, Dezi V, Thomas GH, Willis AE, Lilley KS. Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS). Nat Biotechnol. 2019 Feb;37(2):169-178. doi: 10.1038/s41587-018-0001-2. Epub 2019 Jan 3. Erratum in: Nat Biotechnol. 2019 Jun;37(6):692. doi: 10.1038/s41587-019-0161-8. PMID: 30607034; PMCID: PMC6591131.