Two Novel Trans-Acting Factors Dictate the High Cytoplasmic Stability of β-Globin mRNA

Abstract 644

The efficient accumulation of hemoglobin in mature erythrocytes is critically dependent upon the high stabilities of mRNAs encoding human α- and β-globin proteins. These mRNAs are likely to be stabilized by interactions between one or more trans-acting regulatory factors that target defined cis-acting elements within their 3′UTRs. Several ubiquitous factors that are known to bind to the β-globin 3′UTR (including αCP, PTBP1, and nucleolin) are largely restricted to the nucleus and therefore unlikely to contribute to regulatory processes affecting β-globin mRNA in the cytoplasm. Consequently, we conducted a series of experiments that identify and characterize mRNA-binding factors that dictate the properties of β-globin mRNA in the cytoplasm of erythroid progenitor cells. Using electrophoretic gel mobility shift analyses (EMSA), we defined a characteristic mRNP complex that assembles on the β-globin 3′UTR in cytoplasmic extract–but not nuclear extract–prepared from erythroid K562 cells. This mRNP ‘β-complex’ appears to be erythroid-specific, as it fails to assemble in extracts prepared from non-erythroid HeLa or HEK cells. The 3′UTR binding site for the β-complex was identified using an EMSA-competition approach; remarkably, the target sequence is encompassed within a 12-nt region previously identified as a functional determinant of β-globin mRNA stability in in vivo analyses. Additional experiments fine-mapped the β-complex binding site to a GGGGG pentanucleotide motif within the mRNA-stabilizing region. The functional importance of the pentanucleotide was illustrated by mRNA decay experiments in intact erythroid K562 cells showing that full-length β-globin mRNAs are destabilized by introduction of the same GGGGG->CCGGG mutation that ablates β-complex assembly in EMSA analyses. To identify trans-factors that comprise the β-complex, we performed affinity chromatography using ssDNA probes corresponding to the β-complex binding motif. The native 3′UTR probe retained 42- and 47-kDa proteins, while a probe carrying the CCGGG mutation failed to bind either factor. Subsequent LC/MS/MS analyses identified the two proteins as YB-1 and AUF-1. The identities of these two mRNA-binding factors, which have previously been implicated in the post-transcriptional regulation of heterologous mRNAs, were subsequently confirmed by immunoblot of the protein-DNA complexes. Subsequent analyses suggested a functional role for both factors: EMSA supershift experiments confirmed that YB-1 is a component of the β-complex, and RNA immunoprecipitation analyses demonstrated that both YB-1 and AUF-1 specifically bind to β-globin mRNA in vivo in intact erythroid K562 cells. Collectively, these data identify two novel trans-acting factors that bind to cytoplasmic β-globin mRNA in an erythroid-specific fashion, at a site that dictates its stability in intact cells. We are currently engaged in siRNA knock-down experiments to validate experiments that suggest the importance of these trans-acting factors to the constitutive cytoplasmic stability of β-globin mRNA, as well as structural analyses intended to define RNA-protein and protein-protein interactions that are critical to normal functioning of the β-complex. The results of these experiments have obvious implications for the design of novel therapies for patients with congenital disorders of β-globin gene expression, including sickle cell disease and β thalassemia.