SILAC-based RNA pull-down. Cells are grown in either media with ¹²C₆-Lys/¹²C₆-Arg (light) or ¹³C₆-Lys/¹³C₆-Arg (heavy) (11, 12). RNA is immobilized on paramagnetic streptavidin beads by the S1 aptameric tag, which can be introduced by PCR and subsequent in vitro or in vivo transcription. Bait and control sequence are separately incubated with heavy or light cytosolic extracts, respectively. Both fractions are combined after washing, and RNPs are eluted with biotin. Proteins are measured after tryptic digestion and chromatographic separation of the peptides, which are ionized online by electrospray and analyzed with a high-resolution mass spectrometer. The schematic spectrum presents the ratio distribution between light and heavy forms of the tryptic peptides. Specific interaction partners have a SILAC ratio different from the 1:1 ratios of background binders (6).

2D interaction plots (see Fig. 2) for the identified specific interaction partners for the H4 stem loop, zipcode sequence, and tRNA. (A) The H4 stem loop structure specifically interacts with SLBP as indicated by its high SILAC ratio that is inverted in the cross-over experiment. PGAM5 has a smaller SILAC ratio, but binds reproducibly to the H4 stem loop structure compared with the control sequence. Of 641 identified proteins, these are the only 2 proteins fulfilling the stringent quantitation significance criteria. (B) Only 6 proteins of 781 identified bound specifically to the zipcode sequence. IMP-1, the known interaction partner, is identified as a specific binder in addition to IMP-3, YB-1, CSDA, RBMS1, and C1QBP. (C) RNase P subunits and tRNA enzymatic enzymes are identified as specific binders among 570 identified protein to in vitro-transcribed tRNA. Dermicidine (DCD) is a common contaminant derived from human skin and is therefore unlabeled (very low heavy/light ratio in both pull-downs).

Detection of HuR. (A) 2D plot of RNA pull-down result for HDAC2 mRNA 3′ UTR. The log₂ SILAC ratio of proteins identified with at least 2 unique peptides in each MS run is plotted of the forward pull-down (x axis) against the cross-over pull-down (y axis). Specific interaction partners show inverse ratios between forward and reverse experiment, grouping them into the upper left quadrant. The interval for statistical significance (shaded in the plot) is given by the product of significance in the forward and reverse experiment (P^(for) × P^(rev) < 0.0001) and a minimum SILAC ratio change of a factor 2 (expected experimental variation). Among 552 identified proteins before filtering, the known interaction partner for the HDAC2 3′ UTR fragment (HuR) is represented by a black bullet, having the highest SILAC ratio in the forward and smallest in the cross-over experiment. (B) Western blot confirmation of the MS analysis for HuR. (C) RIP of in vivo cross-linked RNP complexes with antibodies against HUR, MATR3 and isotype control shows enrichment of HDAC2 mRNA at the identified candidates. (D) Differential binding of HuR can be detected by comparing MTA1 3′ UTR wild-type sequence with a deletion variant (SILAC ratio 11.8) or a mutated variant with a destabilized stem loop structure (SILAC ratio 6.5). Between the wild-type sequence and a point mutated variant, no differential binding (SILAC ratio 1:1) is observed. (E) Results of quantitative MS are supported by Western blot analysis. HuR binds to wild-type sequence and the point mutated variant U15G.

DGCR-8 RNA pull-down. (A) MS identification of RPL26/RPL26L as a specific binder to the DGCR-8 wild-type RNA sequence. The doubly-charged tryptic peptide FNPFVTSDR²⁺ was enriched in the heavy form (heavy extract incubated wild-type sequence) in the forward pull-down, whereas the light form was enriched in the reverse pull-down (light extract incubated with wild-type sequence). (B) Immunostaining confirms the binding of RPL26 to the DGCR-8 mRNA fragment observed by quantitative MS. RNA stability in the experiment was monitored by autoradiography of ³²P-labeled RNA from the elution fraction. (C) The tryptic peptide ITMYSSSGK²⁺ of GRSF1 is also enriched specifically with the wild-type sequence in forward and reverse DGCR-8 RNA pull-down. The corresponding SILAC partner was below the signal to noise, demonstrating that the ratio is >1:10 and 10:1, respectively.