A Nucleolin-Binding 3′ Untranslated Region Element Stabilizes β-Globin mRNA In Vivo

Abstract

The normal expression of human ␤ globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with ␣-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing ␤-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the ␤-globin 3 untranslated region (3UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by ␣CP/hnRNP-E, a factor that plays a critical role in stabilizing human ␣-globin mRNA. Mutations within the new determinant destabilize ␤-globin mRNA in intact cells while also ablating its 3UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3UTR-bound nucleolin enhances mRNA stability by optimizing ␣CP access to its functional binding site. This model is favored by in vitro evidence that ␣CP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for ␤-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human ␣-globin mRNA.Erythroid cells accumulate hemoglobin through a process that is critically dependent upon the high stabilities of mRNAs that encode their constituent ␣-and ␤-globin subunits (10, 64). In vivo analyses estimate a half-life for human ␣-globin mRNA of between 24 and 60 h (47,62,63,74), while similar studies with cultured NIH 3T3 and murine erythroleukemia (MEL) cells (2, 36, 42), primary mouse hematopoietic cells (4), and human erythroid progenitors (62, 63) suggest a half-life value for human ␤-globin mRNA that exceeds 16 to 20 h. Globin mRNAs survive, and continue to translate at high levels, for as long as a week following nuclear condensation and extrusion in transcriptionally silent erythroid progenitor cells. As might be anticipated, mutations that impair the normal stabilities of globin mRNAs can severely impact the levels of their encoded proteins. For example, an mRNA-destabilizing mutation reduces the expression of ␣ Constant Spring to less than 2% of normal levels (45,51,80), resulting in a clinically important form of thalassemia characterized by a substantial imbalance in ␣-and ␤-globin chain accumulation (10, 64).The cis-acting determinants and trans-acting factors that participate in regulating ␣-globin mRNA stability have recently been identified, and the relevant molecular mechanisms have been described in detail. Mutational analyses carried out with cultured cells (80, 81) and with animal models (52, 66) clearly demonstrate the importance of the 3Ј untranslated region (3ЈUTR) to the constitutively high stability of ␣-globin mRNA (45). Other studies have mapped this characteristic to a phylogenically conserved, 16-nucleotide (nt) C/U-rich element in this region (39,75,76). The cis-acting pyrimidine-rich element (PRE) assembles an mRNP "␣-complex" that compri...

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“…Nucleolin and heterogeneous nuclear ribonucleoprotein E associate with the 3′ UTR of beta-globin mRNA and extend its half-life. 29 Several RNA-binding proteins (HuR, nuclear factor 90, tristetraprolin, butyrate response factor 1, and K homology-type splicing regulatory protein) preferentially interact with U/AU-rich sequences in the 3′ UTR of the target transcripts and regulate their stability, whereas nucleolin preferentially binds to G-rich sequences. 30 SR proteins also participate in the regulation of mRNA turnover.…”

RNA-binding protein nucleolin in disease

“…Dysregulation of β globin, encoded by HBB mRNA, can result in pathologies such as thalassemia. 48 Nucleolin binds the HBB 3' UTR mRNA and enhances its stability, likely facilitating the binding of hnRNP E. 49 Nucleolin influence on mRNA translation. Nucleolin can also affect the translation of a subset of associated mRNAs.…”

mRNA stability control: a clandestine force in normal and malignant hematopoiesis

“…Several other mRNA targets of nucleolin have been identified. 97,206,207 It will be interesting to see whether the repetoire of nucleolin-bound mRNAs in B-CLL cells is enriched for transcripts coding for antiapoptotic proteins.…”

“…Other proteins implicated in RNA stabilization include RNPC1, 90 the poly (C)-binding protein aCP1, 91 and the ARE-binding proteins NF90, 92,93 and nucleolin. [94][95][96][97] The aCP-binding proteins are important in stabilizing aglobin mRNA and have been reviewed elsewhere. 98 Nucleolin has been investigated extensively for its roles in transcription, splicing and nucleolar function (reviewed by Ginisty et al 99 ) but only a handful of papers have examined it as a regulator of mRNA turnover.…”