Compared to other gene expression regulators such as tran-
scription factors, RNA-binding proteins (RBPs) show higher
expression [18], are able to act both cotranscriptionally and post-
transcriptionally in a concerted manner [19, 20] and are highly
responsive to external cues, such as DNA damage [21]. Therefore,
they represent an important avenue for future research utilizing
systems-wide approaches. An exciting feature of mRNA interac-
tome capture is thus to systematically, quantitatively and differen-
tially analyze the RBPs that change their binding activity upon
certain biological stimuli.
Here, we describe our procedure for isolation and differential
analysis of poly(A)+ RNA-bound proteins. Initially, mammalian
cells in culture are incubated with 4-thiouridine (4SU), resulting
in metabolic labeling of cellular RNA. Following the application of
biological stimulus to one cell population, all samples are exposed
to UV light facilitating the formation of direct protein–RNA cross-
links in living cells and stabilization of protein–RNA complexes
before cell lysis (Fig.1). An important feature of our method is
the usage of a calibrator “heavy” SILAC-labeled lysate [22] that is
spiked into the samples corresponding to both experimental con-
ditions and allows for quantitative comparisons between them.
After a stringent oligo(dT) affinity purification and enrichment of
poly(A)+RNA, the cross-linked proteins are identified and quanti-
fied using mass spectrometry. Afterward, the “heavy” SILAC inten-
sity allows for the correction of between replicate variability due to
separate MS runs, amount of starting material and/or efficacy of
oligo(dT) affinity purifications. We present a detailed experimental
procedure that may be easily applied to different cell culture sys-
tems and stimulus specific responses.2 Materials
2.1 Equipment 1. General cell culture equipment, e.g., incubator and dishes.
- UV cross-linker device (Stratalinker or similar) with UV
365 nm light bulbs. - 15- and 50-mL Falcon tubes.
- Heating block at 80C.
- Magnetic stands for Eppendorf tubes, 15- and 50-mL Falcon
tubes (e.g., Thermo Fisher Scientific DynaMag-2, -15, and -50
magnets). - Sterile cell scrapers.
- Needles and syringes, 23- and 26-G.
- Centrifugal filters with 10,000 MW cutoff and 15-mL volume
(e.g., Millipore. Amicon Ultra-15 Centrifugal Filter).
406 Miha Milek and Markus Landthaler