Table 1.
Sequences of antisense oligonucleotides used in the competitive EMSA against MITF RNA.
Table 2.
Sequences of primers used for amplifying MITF DNA fragments.
Fig 1.
Summary of MITF RNA fragments and their relative binding affinity for CRD-BP.
(A) Two fragments of DNA corresponding to MITF cDNA nts 1330–1550 and 1550–1740 were PCR-amplified with the appropriate primers shown in Table 2. (B) Four fragments of 3’ end truncated DNA corresponding to MITF cDNA nts 1550–1740 were generated as in (a) using the appropriate primers listed in Table 2. (C) Four fragments of 5’ end truncated DNA corresponding to MITF cDNA nts 1581–1740 were generated using the appropriate primers shown in Table 2. 32P] RNA was prepared from each of the above DNA template and analyzed by electrophoretic mobility shift assay (EMSA) for CRD-BP binding shown in Fig 2. (++++), (+++), (++), and (+) indicate ~90%, ~70%, ~40% and ~10% binding respectively. (+/-) and (-) indicate weak and no protein-RNA complex formation respectively.
Fig 2.
Mapping the CRD-BP binding site on MITF RNA using electrophoretic mobility shift assay.
(A) Two fragments (nts 1330–1550 and 1550–1740) of [32P] MITF RNA (13 nM) covering nts 1330–1740 of MITF cDNA were incubated with various concentrations of recombinant CRD-BP as shown. [32P] c-myc RNA nts 1705–1886 was used as a positive control. (B) Four 3’ end truncated fragments (nts 1550–1709, 1550–1609, 1550–1619, and 1550–1589) of [32P] MITF RNA covering nts 1550–1740 of MITF cDNA were incubated with various concentrations of recombinant CRD-BP and analysed using EMSA as shown. The [32P] MITF RNA nts 1550–1740 was used for comparison. (C) As above, four 5’ end truncated [32P] MITF RNAs (nts 1581–1740, 1621–1740, 1671–1740, and 1701–1740) were assessed for their ability to bind CRD-BP. Samples were electrophoresed in a 4% native polyacrylamide followed by autoradiography. In each gel, free RNA and CRD-BP-bound RNA are indicated.
Fig 3.
Assessing the role of nucleotides 1621–1669 of MITF RNA in binding CRD-BP.
Electrophoretic mobility gel-shift assay on the binding of purified recombinant WT CRD-BP to MITF RNA 1550–1740, MITF RNA 1550–1740 (Δ1621–1669) and MITF RNA 1621–1669. Various concentrations of CRD-BP, as shown, were incubated with 40 nM [32P] MITF RNA. Arrows indicate the free RNA and the brackets indicate CRD-BP-bound RNA. Dissociation constant (Kd) for CRD-BP to bind MITF RNA 1550–1740 and MITF RNA 1550–1740 (Δ1621–1669) are shown at the bottom of the image. The Kd for binding to MITF RNA 1621–1669 could not be determined.
Fig 4.
Binding profiles of CRD-BP and its point mutation variants on MITF RNA.
(A) Electrophoretic mobility gel-shift assay on the binding of purified recombinant WT CRD-BP and its point mutation variants to [32P] MITF RNA nts 1550–1740 (191 nts). Various concentrations of proteins, as indicated, were incubated with 40 nM of the radiolabeled MITF RNA. Samples within each panel indicate results from the same gel generated from the same experiment. Data shown are representatives from at least three experiments using at least two separately prepared recombinant proteins. The positions of protein-RNA complexes (Bound) and free RNA are indicated. (B) A summary of dissociation constants (Kd) of the WT CRD-BP and its variants. The Kd values were taken from saturation binding curves (n = 4). The single asterisk indicates that the p-value is less than 0.05 based on Student’s t-test in comparing to the Kd of WT CRD-BP. The double asterisks indicate that there was significant reduction and hence the Kd could not be determined.
Fig 5.
Predicted RNA secondary structure of MITF RNA and target sites of antisense oligonucleotides.
The predicted secondary structure of MITF RNA (nts 1550–1740) was generated using the MFOLD program [27]. As shown, the solid lines indicate complimentary regions where each of the 8 MHO antisense oligonucleotides hybridizes to. The number within the bracket indicates the specific nucleotide sequences where the MHO antisense oligonucleotides target.
Fig 6.
Inhibition of the CRD-BP-MITF RNA interaction by antisense oligonucleotides.
(A) Purified recombinant CRD-BP (2000 nM) was incubated with [32P] MITF RNA nts 1550–1740 in the presence of antisense oligonucleotides MHO-1 to MHO-8 at 10- or 50-fold molar excess. As a positive control, 50-fold molar excess of unlabelled MITF RNA nts 1550–1740 (lane 3) was used. (B) As in (A), 5-, 25-, and 50-fold molar excess of antisense oligonucleotides MHO-1, MHO-7, MHO-8, GLI1 RNA nts 346–382 and DD7 oligonucleotide was competed with [32P] MITF RNA nts 1550–1740 for binding to CRD-BP. (C) A wider concentration range of antisense oligonucleotides MHO-1 and MHO-7 at 1- to 500-fold molar excess were used in the competitive EMSA against [32P] MITF RNA nts 1550–1740. Samples were electrophoresed in a 4% native polyacrylamide followed by autoradiography. In each gel, free RNA and CRD-BP-bound RNA are indicated. (D) The average percentage of bound complex, taken from (C) and two other separate experiments is expressed on the graph as shown.
Fig 7.
Assessing the binding of MHO-1 and MHO-7 to MITF RNA and CRD-BP.
(A) RNase protection assay to assess the binding of MHO-1 and MHO-7 antisense oligonucleotides to MITF RNA 1550–1740. 32P-MITF RNA 1550–1740 was incubated with tRNA, MHO-1 or MHO-7 overnight at 42°C as described in Materials and Methods. The reactions were then subjected to RNase H treatment, precipitated, and ran on 8% denaturing polyacrylamide gel as shown. 32P-MITF RNA fragments which were not digested by RNase H are shown by arrows. The 75 nts size KRAS RNA on lane 4 was used as a marker. The schematic diagram on the right illustrates the action of RNase H and the expected RNA fragments generated upon hybridization of MHO-1 and MHO-7 to 32P-MITF RNA 1550–1740. (B) Fluorescence polarization assays to assess the binding of MHO-1 and MHO-7 to MITF RNA (left panel) and CRD-BP (right panel). Ten nM of fluorescein-labeled MHO-1 and MHO-7 AONs as well as fluorescein-labeled CD44 RNA were incubated with an increasing concentration of MITF RNA (0–1000 nM) (left panel) or CRD-BP (0–1000 nM) (right panel) as described in Materials and Methods. The error bars are S.E. and the results are representative of two separate experiments.
Table 3.
A summary of the dissociation constants (Kd) of WT CRD-BP in comparison with its KH variants for binding to several RNA targets.