Dealing with Experimental Noise
The applicability of CLIP-derived procedures is contingent to the extent
of their background. A general assumption of these protocols is that
random binding is hampered by the harsh washing steps succeeding
immunoprecipitation. Moreover, electrophoretic resolution of the RNPs
under study ensures an additional enrichment step with respect to
adventitiously co-partitioned RBPs. Preferred control samples have
normally been acquired from cells where the gene encoding the RBP of
interest is knocked out, untagged or which have not been UV treated (Fig
2B-C) 22,23,27,37. In principle, these samples are not
expected to produce any noticeable signal upon SDS-PAGE visualisation.
Consequently, libraries emerging from the sequences in the areas
corresponding to the expected migration of the RNP under study should
contain 100-fold less unique cDNAs than those resulting from fully
treated samples 37. Nonetheless, meta-analysis of 30
published CLIP datasets identified widespread and replicable background
reads across most of them 38. To quantify technical
background, prior work sequenced libraries proceeding from RNA-protein
adducts that did not migrate with the duplexes of interest (Fig. 2C)38. Adjusting output signals to those originated by
background characterisation was shown to dramatically improve the
identification of RNA recognition sequences 38. Based
on relevant control samples, background correction can indeed
statistically disregard binding events resulting from non-specific
transcript cross-linking to incidentally proximal RBPs of interest,
co-purified RNPs and RNA aptamers adopting an epitope-mimicking
conformation.
A number of CLIP-related approaches have been developed that have been
specifically designed to further improve signal-to-noise ratios. These
methods take full advantage of the fact that cross-lined RNPs can
withstand highly denaturing purification conditions. These techniques
include cross-linking and analysis of cDNAs (CRAC), ultraviolet
crosslinking and affinity purification (uvCLAP) and denaturing CLIP
(dCLIP). All of these protocols employ (tandem) affinity-based
purification to recognise specific epitope tags that are compatible with
denaturing purification conditions and/or strong detergents (e.g.,
HIS-tag and streptavidin-binding peptides sequences; Fig. 1)23,39,40. Admittedly, epitope tagging can alter the
stability, expression, and RNA-binding capacity of the RBP of interest.
Advantageously, however, specific tag recognition-based partition spares
the need for high-quality antibodies, which are not readily available
for less well studied model organisms. Accordingly, these approaches can
considerably reduce background reads in resulting cDNA libraries. This
property also makes them good alternatives to inspect scarcer RNPs,
namely yeast pre-ribosomal complexes or those encompassing
non-professional RBPs 2,23.