Conformational-dependent and independent RNA binding to the fragile x mental retardation protein

Abstract

The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

Figure 1

NaCl blocks nonspecific binding of FMRP with avidin. (a) ³⁵S-FMRP was bound to SoftLink resin in 1x Schaeffer binding buffer supplemented with 0 mM, 125 mM, 250 mM and 375 mM NaCl, lanes 2–5, respectively. Bound ³⁵S-FMRP was recovered, resolved by SDS-PAGE and subject to autoradiography. Lane 1 shows the amount of ³⁵S-FMRP input into the assay. (b) ³⁵S-FMRP was bound to SoftLink resin in 1x Schaeffer binding buffer supplemented with 0.05 mg/mL, 0.1 mg/mL, 0.15 mg/mL, and 0.25 mg/mL tRNA, lanes 2–5, respectively. Bound ³⁵S-FMRP was recovered, resolved by SDS-PAGE and subject to autoradiography. Lane 1 shows the amount of ³⁵S-FMRP input into the assay. The asterisk marks a ³⁵S-truncation product produced by transcription/translation.

Figure 2

FMRP interacts weakly with annealed BC1 RNA. Equal amounts of ³⁵S-FMRP was titrated with various amounts of (a) unannealed biotinylated-BC1 RNA, (b) annealed biotinylated-BC1 RNA, and (c) unannealed biotinylated-eEF-1A RNA, as indicated. Bound (B) and unbound (U) ³⁵S-FMRP was recovered, resolved by SDS-PAGE and subject to autoradiography. Nonspecific binding to the SoftLink avidin resin is shown in the -RNA lanes. (d) Graphical analysis of the above data for 4 reactions per concentration. (e) Binding of annealed or unannealed eEF1A RNA (60 nM) to ³⁵S-FMRP. The percent binding corrected for background of 4 reactions per RNA type is plotted (P = .45 by ANOVA).

Figure 3

Annealed BC1 RNA and unannealed BC1 RNA differ structurally. (a) Secondary structure model of BC1 RNA from M-fold. Black arrows indicate potential RNase VI cleavage sites (both sides of the stem); gray arrows mark potential RNase A cleavage sites. (b) Serial treatment of annealed and unannealed full-length BC1 RNA with RNase A starting at 1 ng as indicated. (c) Ten-fold serial treatment of annealed and unannealed full-length BC1 RNA with RNase VI starting with 0.1 units/μL (upper panel), or 0.5 units/μL (lower panel) as indicated. RNA was visualized by ethidium bromide staining. Arrows mark the major conformers that can be resolved in this system. Lane M shows 100 bp molecular weight markers.

Figure 4

Functional dissection of structural elements in the 5′ end of BC1 RNA. (a) Ten-fold serial treatment of annealed and unannealed 5′BC1-75 RNA with RNase VI starting at 0.1 units/μL (upper panel) or 0.5 units/μL (lower panel). Arrow marks the major conformer. Lane M shows 100 bp molecular weight markers. (b) Secondary structure model of 5′BC1-75 RNA from M-fold.

Figure 5

Functional dissection of structural elements in the 3′ end of BC1 RNA. (a) Ten-fold serial treatment of annealed and unannealed 3′BC1-60 RNA with RNase VI starting at 0.1 units/μL (upper panel) or 0.5 units/μL (lower panel). Arrow marks the major conformer. Lane M shows 100 bp molecular weight markers. (b) Secondary structure model of 3′BC1-60 RNA from M-fold.

Figure 6

Interaction of FMRP with the 5′ and 3′ ends of BC1 RNA. (a) ³⁵S-FMRP was incubated with 2 μM annealed full-length BC1 RNA, or annealed versions of the first 75 b of BC1 RNA (5′BC1-75), the first 60 b of BC1 RNA (5'BC1-60) the last 60 b of BC1 RNA (3′BC1-60) and an 85 b transcript containing the HIV1 TAR hairpin. The percent binding corrected for background of 6 reactions per protein is plotted. (b) Secondary structure model of 5′BC1-60 RNA from M-fold. (c) Secondary structure model of the HIV1 TAR RNA from M-fold; the 27 b leader sequence has no effect upon the folding.

Figure 7

Annealed BC1 RNA binds pleiotropically to the FXRPs. (a) Equimolar amounts of ³⁵S-FMRP, ³⁵S-FMRP_15c, ³⁵S-FXR1P, ³⁵S-FXR2P, ³⁵S-eIF4A, ³⁵S-FMRP₂₈₀, ³⁵S-FMRP₂₀₄, and ³⁵S-Luciferase were incubated with 2 μM annealed BC1 RNA. The percent binding corrected for background of 6 reactions per protein is plotted. The relative levels of FMRP and FMRP_15c, FMRP and FXR1P and FMRP and FXR2P were not significantly different (P = .17, P = .67 and P = .27, resp., ANOVA). However, the relative levels of FMRP and eIF4A and FMRP and NTD₂₀₄ were significantly different (P = .038 and P = .007, resp., ANOVA). Since no binding above background was observed for NTD₂₈₀ and Luciferase relative differences were not assessed. (b) Pull-down assays between ³⁵S-luciferase and poly (rG), poly (rA), eEF1A RNA (90 nM) and annealed BC1 RNA (5 μM) showing the unbound (U) and bound (B) fractions. Binding in the absence of RNA is shown for the negative control. (c) Pull-down assays between ³⁵S-NTD₂₈₀ or ³⁵S-NTD₂₀₄ and poly (rG), poly (rA), poly (rI : rC) and annealed BC1 RNA (5 μM) showing the unbound (U) and bound (B) fractions. Binding in the absence of RNA is shown for the negative control.