The filter-binding assay was performed by a modified method described by Wong et al. (31) . Briefly, radiolabeled, in vitro transcribed RNA at a concentration of $10 nM was heat denatured, allowed to refold and incubated for 15 min in room temperature in binding buffer (20 mM Tris, pH 7.9, 100 mM KCl, 2.5 mM MgCl 2 ) containing heparin (5000 U/ml, Sigma) and 160 U of RNAse inhibitor (RNase OUT, Invitrogen) with purified Hax-1 protein. The reaction mix was loaded onto a presoaked nitrocellulose membrane (0.45 mm, Osmonic Inc.) on top of a nylon membrane (Hybond-N, Amersham) and filtered under pressure in a slot-blot apparatus. Following filtration, each filter was dried and quantitated on a PhosphorImager (BioRad) using QuantityOne software. Dissociation constants (K d ) for the RNA-protein complexes were obtained by fitting the empirical data to a sigmoidal curve by nonlinear regression analysis, using Maxima 5.4. Fitting was performed in respect to dimer concentration calculated as a function of the total protein concentration, as in (30) .