Structural determinants of human zeta-globin mRNA stability


The normal accumulation of adult alpha and beta globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic zeta globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis.

Recently, though, zeta globin has been recognized as a potential therapeutic for alpha thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous zeta-globin genes in definitive erythroid progenitors.

Methods:
The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of zeta-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3[prime]UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure.

Results:
We specify a tetranucleotide 3[prime]UTR motif that is required for the high-level accumulation of zeta-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the zeta-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify.

Additional studies demonstrate that the zeta-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult beta-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development.

Conclusions:
These data demonstrate a mechanism for zeta-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in alpha thalassemia and sickle-cell disease.

Author: Zhenning HeDecheng SongSebastiaan van ZalenJ Eric Russell
Credits/Source: Journal of Hematology Oncology 2014, 7:35

Published on: 2014-04-21

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