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. 2014 Jul 1;3:e02637. doi: 10.7554/eLife.02637

Figure 2. In vitro characterization of the binding preferences of the SCML2 RBR.

(A) EMSA with 2 (+) and 4 (++) pmol GST-fused RBR and 520 fmol HOTAIR RNA 1–300, 690 fmol nucleosomes, or 275 fmol dsDNA encoding HOTAIR1–300. 5.2 pmol GST were used as a control. Complexes were separated on native gels and detected with SYBR gold stain. Data are representative of ≥4 experiments. (B) EMSA with 3.4 pmol GST–RBR and 520 fmol of labeled HOTAIR RNA was performed as in (A) with the addition of, from left to right, 260, 520, and 1040 fmol of unlabeled HOTAIR RNA, nucleosome particles, dsDNA, and 601 RNA. Bands were visualized by autoradiography. Data are representative of ≥4 experiments. (C) EMSA of RBR with nucleosomes and labeled HOTAIR RNA. Assay was performed as in (B) varying the amount of labeled RNA. Nucleic acid stain (top) and autoradiography (bottom) are shown. The asterisk indicates the putative ternary complex.

DOI: http://dx.doi.org/10.7554/eLife.02637.004

Figure 2.

Figure 2—figure supplement 1. Additional EMSAs.

Figure 2—figure supplement 1.

(A) Assay performed as in Figure 2A but using the MBT–DUF fragment of SCML2. Data shown is representative of ≥4 experiments. (B) Assay performed as in Figure 2B but using the MBT–DUF fragment of SCML2. Data shown is representative of ≥4 experiments.
DOI: http://dx.doi.org/10.7554/eLife.02637.005
Figure 2—figure supplement 2. Ternary complex EMSAs and pull-downs.

Figure 2—figure supplement 2.

(A) Models for the interpretation of EMSA results shown here and in Figure 2. (I) The multiple shifted bands can be explained by multiple copies of SCML2 fragments binding to a single RNA molecule or nucleosome. (II) Alternatively, multiple copies of SCML2 can establish bi- or poly-valent interactions with the nucleic acids. (III) Finally, results shown in Figure 2 support the possibility that a monomer of RBR establishes multiple simultaneous contacts with the RNA and nucleosomes. (B) Assay performed as in Figure 2C but this time after removing the GST moiety from the RBR fragment to exclude the possibility of GST-induced dimerization. (C) Biotinylated nucleosomes were incubated with GST–RBR and various amounts of HOTAIR RNA and pulled-down with streptavidin. Western blots for histone H3 (top), GST–RBR (middle), and SYBR stain for HOTAIR (bottom) are shown. Data are representative of two experiments. (D) EMSA with 690 fmol nucleosomes and HOTAIR RNA 1–300 (260, 520, 1040 fmol). Complexes were separated on native gels and detected with SYBR gold stain.
DOI: http://dx.doi.org/10.7554/eLife.02637.006