Nucleolin-Mediated Stabilization of Human β-Globin mRNA.

The normal expression of human β globin is critically dependent upon the high stability of its encoding mRNA. The mechanism that protects β-globin mRNA from premature degradation--including the positions of cis-acting stability determinants, the identities of relevant trans-acting factors, and the processes through which they interact--are poorly understood. We have designed and executed a series of experiments that detail the critical importance of the 3′UTR to the high constitutive stability of β-globin mRNA. To identify mRNA-stability determinants in this region, we constructed a wild-type β-globin gene (β^WT), as well as 17 derivative genes containing site-specific 3′UTR hexanucleotide substitutions (β^MUT1-β^MUT17), each under the transcriptional control of a tetracycline-response element (TRE). In cultured cells that express a corresponding transcriptional transactivator, the expression of TRE-linked β^WT and β^MUT genes can be rapidly silenced by adding tetracycline to the culture medium, permitting the stabilities of the cognate mRNAs to be established using a transcriptional chase approach. Two of the 17 mRNAs, carrying adjacent hexanucleotide substitutions (β^MUT12 and β^MUT13), were destabilized in intact HeLa cells, identifying a sequence that is critical to β-globin mRNA stability. Three potentially important trans-acting factors that bind to this region were subsequently isolated using an in vitro affinity-enrichment method. One of the proteins was unequivocally identified by mass spec analysis to be nucleolin, a ubiquitous nuclear-cytoplasmic factor that exhibits RNA helicase activity and is reported to stabilize several non-erythroid mRNAs. A link between this factor and β-globin mRNA stability was provided by in vitro studies demonstrating that purified nucleolin binds tightly to the β^WT 3′UTR but poorly to both β^MUT12 and β^MUT13 3′UTRs. This result was validated by RNA-immunoprecipitation (RIP) analyses confirming a strong interaction between nucleolin and β^WT mRNA in intact cells that is fully ablated by MUT12 or MUT13 hexanucleotide substitutions. The critical importance of nucleolin binding to the stability of β-globin mRNA may relate to a stem-and-loop motif within its 3′UTR that is predicted by mRNA-folding algorithms. This structure contains the nucleolin-binding site on its right half-stem, opposite a putative binding site for αCP, a 34 kDa factor that stabilizes α-globin mRNA, on the left half-stem. Surprisingly, recombinant αCP displays a low affinity for the full-length β-globin 3′UTR, while binding avidly to the isolated left half-stem as well as to full-length β-globin 3′UTRs that contain stem-disrupting mutations. These results indicate that high-order structures within the β-globin 3′UTR, if permitted to form, may interfere with αCP function in vivo. Based upon our studies, we suggest that nucleolin binding is required to relax a highly stable stem-and-loop motif within the β-globin 3′UTR, exposing a functional binding site for the mRNA-stabilizing factor αCP. Thus, we identify a cis-element and a specific trans-acting factor that participate in stabilizing β-globin mRNA, and suggest a mechanism through which they are likely to act in vivo. The full elucidation of this process will clearly benefit the design of therapeutic transgenes for individuals with β-globin gene defects, and may additionally facilitate the conception of novel therapies intended to differentially regulate the stabilities of β^S- and γ-globin mRNAs in individuals with sickle cell disease.