Abstract 4059
Poster Board III-994
Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease.
Disclosures:
No relevant conflicts of interest to declare.
Cis-regulatory elements explain most of the mRNA stability variation across genes in yeast
