Dealing with Noise
In contrast to organic phase extraction approaches, silica-based strategies typically yielded higher numbers of significantly enriched RNA-binders. In fact, whereas prior reports had estimated RBPs to conform around a tenth of eukaryotic and bacterial proteomes7,14, silica-based findings consistently record portions between 20-30% 2,19. It is plausible that spurious proteins co-purify with RNPs if, like histones or nucleotide-binding enzymes, they bind to DNA or short nucleotides20. However, many of these proteins were also abundantly detected in datasets stemming from organic phase separation experiments, which cannot detect RNA-binding in transcripts shorter than 30 nucleotides in length 15. Regardless, we strongly recommend testing a range of UV irradiation treatments and selecting the lowest possible dose when studying protein-RNA interactions19: high levels of UV not only cause substantial protein and RNA degradation 2,21, but also can cross-link proteins to DNA 20. Thus, treating lysates with DNase is also advisable. Notably, although 2C and TRAPP indeed generate larger pools of statistically enriched proteins, they generally require a larger number of input cells to do so. This constitutes a technical cost for the poorer binding capacity of silica-based capture compared to self-contained phase separation systems.
Despite the obvious methodological differences as well as the advantages and shortcomings which they prompt, silica-dependent and organic phase separation-based produced remarkably similar results for S. aureus 2. Together with individual validation of the RNA-interaction status of some novel RBPs, this first direct comparison underscored that both approaches are fit for holistic interrogation of microbial RBPomes. In fact, the combination of both strategies could improve the current individual performance of both methods. For instance, in the XRNAX protocol, mammalian RNPs were coarsely partitioned from other cellular components using phenol extraction and further enriched using a silica-conjugated platform, which contributed to reduce noise (Fig. 1) 16.