This proposal extends an ongoing research program that is committed to defining the latent therapeutic value of epsilon-globin gene reactivation in adults with disorders in beta-globin expression. The potential benefit of epsilon-globin in these individuals derives from its incorporation into Hb heterotetramers that display physiologically appropriate O2-binding and antisickling properties. As post-transcriptional mechanisms play essential roles in the regulated expression of other globin genes, it is likely that similar processes affecting the stability and translational efficiency of epsilon-globin mRNA are equally important to its expression. Pilot studies carried out in intact animals and in vitro implicate specific cis sequences and defined trans-acting factors as participants in regulated epsilon-globin mRNA stability, several of which are highly similar to well-described determinants of adult-stage beta-globin mRNA stability. The current proposal extends these pilot studies in three Specific Aims that investigate independent aspects of regulated epsilon-globin mRNA stability.
The Aims utilize well-established techniques that draw upon the applicant's experience, as well as several novel methods that have been validated in preliminary studies.
Aim I will define specific cis-acting elements that dictate the stability of epsilon-globin mRNA, through novel cell culture analysis of epsilon-globin mRNA variants containing defined site-specific 3'UTR mutations. The physiological importance of these determinants will subsequently be validated in a well-established transgenic mouse model system.
Aim II will identify the specific cytoplasmic factors that effect the characteristic stability of epsilon-globin mRNA and may also participate in co-regulating the stabilities of beta- and gamma-globin mRNAs. These experiments will be carried out in vitro and in vivo using familiar analytical methods.
Aim III will investigate the effect of regulated epsilon-globin mRNA stability on crucial physiological and molecular processes, including its developmental stage-restricted expression and its translational efficiency in definitive erythroid cells. These experiments will capitalize on several unusual but highly informative methods that have been developed for this specific purpose in the applicant's laboratory. The results from all three Aims will integrate to provide a solid understanding of the fundamental molecular processes regulating the stability of epsilon-globin mRNA, the manner in which these processes may affect the stability of other globin mRNAs, and the physiological consequences--both desirable and undesirable--that result from their targeted dysregulation. The proposed research comprises a crucial step in evaluating the promise of developmentally silenced globin genes for individuals with beta-thalassemia and sickle cell anemia.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL061399-08
Application #
7054071
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Evans, Gregory
Project Start
1999-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
8
Fiscal Year
2006
Total Cost
$309,551
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Safo, Martin K; Ko, Tzu-Ping; Schreiter, Eric R et al. (2015) Structural basis for the antipolymer activity of Hb ?2?(s)2 trapped in a tense conformation. J Mol Struct 1099:99-107
van Zalen, Sebastiaan; Lombardi, Alyssa A; Jeschke, Grace R et al. (2015) AUF-1 and YB-1 independently regulate ?-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein. Mech Dev 136:40-52
He, Zhenning; Song, Decheng; van Zalen, Sebastiaan et al. (2014) Structural determinants of human ?-globin mRNA stability. J Hematol Oncol 7:35
Safo, Martin K; Ko, Tzu-Ping; Abdulmalik, Osheiza et al. (2013) Structure of fully liganded Hb ?2?2s trapped in a tense conformation. Acta Crystallogr D Biol Crystallogr 69:2061-71
Abdulmalik, Osheiza; Lombardi, Alyssa A; Russell, J Eric (2012) A reverse time-course method for transcriptional chase analyses of mRNA half-lives in cultured cells. PLoS One 7:e40827
van Zalen, Sebastiaan; Jeschke, Grace R; Hexner, Elizabeth O et al. (2012) AUF-1 and YB-1 are critical determinants of ýý-globin mRNA expression in erythroid cells. Blood 119:1045-53
Manning, James M; Popowicz, Anthony M; Padovan, Julio C et al. (2012) Intrinsic regulation of hemoglobin expression by variable subunit interface strengths. FEBS J 279:361-9
Liu, Xingge; Jiang, Yong; Russell, J Eric (2010) A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR. Thromb Res 126:130-6
Manning, Lois R; Popowicz, Anthony M; Padovan, Julio et al. (2010) Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces. Protein Sci 19:1595-9
Liu, X; Russell, J E (2010) Cytoplasmic stabilities of 3'UTR-polymorphic prothrombin mRNAs. J Thromb Haemost 8:2580-3

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