Toward a consensus on the binding specificity and promiscuity of PRC2 for RNA

Abstract

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Early works suggested binding specificity of PRC2 to certain long non-coding RNAs for recruitment to chromatin. More recent studies provided evidence both in favor and against this idea. Here, we bridge the two existing models of PRC2-RNA interaction. RepA RNA is a good binding partner for PRC2, while multiple non-relevant RNAs, including bacterial mRNAs, also bind PRC2; Kds depend to some extent on the experimental conditions. Human and mouse PRC2 have broadly similar RNA-binding properties in vitro. Examination of evidence supporting an existing model for site-specific recruitment of PRC2 by a well-defined RNA motif in cells reveals that results are PRC2 independent. We conclude that promiscuous and specific RNA-binding activities of PRC2 in vitro are not mutually exclusive, and that binding specificity in vivo remains to be demonstrated.

Figure 1. Adequate control for RNA length is necessary to assess RNA binding specificity

EMSA performed as previously described (Cifuentes-Rojas et al., 2014), using human PRC2 5m. (A) Large variation in affinity is observed when non-specific RNAs are shorter than the canonical RNA for which they are controlling. Same RNAs as used in (Cifuentes-Rojas et al., 2014): RepA I-IV, 434 bases long RNA, including all tandem repeats from the repeat A region within mouse RepA RNA; MBP 1-300, 300 bases from the 5’ end of MBP mRNA from E. coli ; P4P6, 159 bases long RNA from Tetrahymena group I self-splicing intron. (B) Experiment repeated with RepA and size-matched RNAs, serving as an adequate control for RepA length, including 434 bases from the 5’ ends of protein coding mRNA sequences from bacterial β-lactamase, E. coli maltose binding protein (MBP), Firefly luciferase and glutathione S-transferase (GST) from Schistosoma japonicum (blood fluke). Dissociation constants and Hill coefficients are indicated. Dashed boxes indicate the protein concentrations required for shifting half of the radiolabeled RNA, namely K_d, as identified by densitometry. Error bars within binding curves and standard deviations for binding constants represent three independent experiments, performed on different days. K_d stands for dissociation constant and n_H for Hill coefficient. Binding specificity represented as fold-change between dissociation constants that were observed for RepA and non-specific RNAs, as previously defined (Johansson et al., 1998). Same values used to calculate the corresponding differences in Gibbs free energy (ΔΔG). See also Figure S1 and Table S1.

Figure 2. Complete binding curves of mouse RepA I-IV in the presence of different concentrations of human PRC2 5m, human PRC2 4m and mouse PRC2 4m, in comparison to a non-relevant RNA control of the same length

(A) Binding and EMSA conditions are as previously described (Cifuentes-Rojas et al., 2014). To control for RepA I-IV length, a 434 bases long RNA comprising the 5’ end of the coding sequence of the non-relevant bacterial beta-lactamase mRNA (β-lactamase 434) was used. Error bars within binding curves and standard deviations within the table represent two to three independent experiments, performed on different days. (B) Filter Binding assay performed as previously described (Cifuentes-Rojas et al., 2014) for the same RNAs and PRC2s. Specificity defined as in Figure 1. Error bars within binding curves and standard deviations within the table represent at least three independent experiments, performed on different days. See also Figure S1 and Table S1.

Figure 3. Apparent RNA binding affinity depends on experimental conditions

(A) RepA I-IV and P4P6 RNAs, as previously used (Cifuentes-Rojas et al., 2014), were subjected to a qualitative binding experiment, where the RNA folding protocol and PRC2-RNA incubation were performed according to either (Davidovich et al., 2013) or (Cifuentes-Rojas et al., 2014). Each sample was split into two and each portion loaded on a non-denaturing gel, buffered either with TBE or THEM. Agarose gel density and running conditions were as published within these references. (B) Same RNAs and PRC2, as in Figure 1, were subjected to quantitative binding experiments using binding buffer including 50 mM Tris HCl, pH 7.5 (@ RT), 100 mM KCl, 2.5 mM MgCl₂, 0.1 mM ZnCl₂, 2 mM 2-mercaptoethanol, 0.1 mg/ml BSA, 0.1 mg/ml yeast tRNA (Sigma R5636), 0.05 % v/v NP40 and 5 % v/v glycerol. Samples were loaded on a 0.7% (w/v) agarose gel, buffered with TBE, and EMSA carried out as previously described (Davidovich et al., 2013). Error bars within binding curves and standard deviations within the table represent three independent experiments, performed on different days. See also Figures S2 and S3 and Table S1.