A challenge of CLIP procedures is, again, their dependence on UV
exposure to generate RNA-protein adducts. As outlined above, in
vivo UV radiation is particularly inefficient: it usually takes minutes
to complete and only about 1-5% of RNPs of interest are cross-linked28. Hence, some CLIP variants often require relatively
large number of cells. Systematically poor cross-linking performance
could be attributed to the impossibility of creating adducts with some
amino or intrinsically limited UV penetration in certain cell types or
growth media 2,15. Recently, we developed a
much-improved UV cross-linker (Vari-X-linker; UVO3
1,21) that greatly increases the cross-linking efficiency in actively
growing cells during markedly shorter time spans (seconds)1,21. However, the fastest and most efficient way to
cross-link proteins to RNA is using UV lasers 29,
although setting up such a system can be prohibitively expensive.
Another major advantage of these rapid cross-linking devices is that
they enable the monitoring of very dynamic changes in protein-RNA
interactions, such as those occurring during stress responses,
at
high temporal resolution 1,21,29,30.
To quantify
RNA-protein
cross-linking efficiencies, most protocols attach radioactive phosphates
or fluorescently labelled oligonucleotides to 5’ ends of the
cross-linked transcripts. The RNP complexes are subsequently resolved by
denaturing PAGE (Fig. 2) and the RNA can then be visualised by
autoradiography or fluorescent imaging. Recently, we performed such
radiolabelling analyses on a variety of RBPs identified in our S.
aureus RBPome analyses. While the vast majority of the proteins tested
detectably cross-linked to RNA, we learned that only for those proteins
for which we observed very strong radioactivity signals (1 to 3-hour
exposure of phosphoimager screen/film) were we able to obtain
high-complexity complementary DNA (cDNA) libraries. It is also important
to point out that the radiolabelling assay can generate false positive
signals: radioactive labelling of cross-linked RNA involves an
incubation step with T4 polynucleotide kinase (T4 PNK). However, it is
possible that during this reaction the RBP of interest becomes
radiolabelled by autophosphorylation or co-purified host kinases31. Consequently, we would always advise including
control reactions with non-irradiated cells or leaving out PNK
altogether (Fig. 2A).
As well as informing the choice of UV exposure, preliminary signal
evaluation provides a strategy by which to filter out proteins with no
or little RNA-binding activity. As outlined above, UV can also
cross-link proteins to DNA 20. Accordingly, we
strongly recommend performing control experiments where the cross-linked
RNPs are incubated with increasing concentration of RNase I/A or DNase
I. If the purified RNP indeed contains cross-linked RNA, higher
concentrations of RNase should reduce the smearing of the bands
corresponding to RNPs (Fig. 2B; PNPase). Alternatively, the intensity of
those containing DNA-protein duplexes would only be resolved by DNase
digestion. We also found that not all cross-linked proteins, such as the
superoxide dismutase SodM, respond to either DNase or RNase treatment
(Fig. 2B). This suggests that these proteins generally bind short
nucleic acid fragments, which will make the library preparation steps
more challenging.