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.