In order to confirm the results of the 3-hybrid screen, analysis of in vitro binding was carried out using purified (14) . The free energy increment for the two structures varies considerably, and amounts to À14.1 for the M2 hairpin and À3.6 for the mutant. Presence of the hairpin-containing 3 0 UTR confers mRNA instability but also enhances expression at the protein level. Disruption of the hairpin prevents mRNA degradation, but a mutation-containing construct still slightly enhances expression at the protein level. Constructs used for transfections: control vector (pCMLuc), hairpin-containing construct (pCMLucH) and mutated hairpin (pCMLucHmut). mRNA levels were quantitated for each construct by RQ-PCR in cDNA isolated from transfected cells (8 independent transfections), in triplicate, normalized with respect to Renilla luciferase mRNA levels. Firefly luciferase levels were assessed for the same eight transfections (in six repeats) and normalized with respect to Renilla luciferase levels, which served as a control of transfection efficiency. Statistical significance is reported as P-value: Ã P50.01, ÃÃ non-significant. UV-cross-linking was performed using the purified recombinant Hax-1 and in vitro transcripts of the hairpin-containing region (H), a control antisense transcript of the same region (A), and the mutated transcript (Hmut). Bovine serum albumin and purified thioredoxin served as controls for interaction specificity. Hax-1 demonstrated a specific interaction only with the hairpin-containing RNA, and this transcript did not interact with either BSA or thioredoxin ( Figure 6) . The cross-linked band migrated at a molecular weight of $100 kDa, which corresponds to a recombinant Hax-1 dimer (the recombinant protein has a higher molecular mass than endogenous Hax-1 [35 kDa], due to its fusion with the thioredoxin sequence). The protein dimer is likely formed during the UV-cross-linking procedure, as has been previously documented (34) . No band corresponding in size to monomeric recombinant Hax-1 (ca. 50 kDa) was observed, which indicates that the protein interacts with RNA exclusively in the form of a dimer.