The normal expression of human ? globin is critically dependent upon the unusually high stability of its encoding mRNA. The current proposal is designed to specify the fundamental features of a newly described molecular mechanism that appears to dictate the constitutive stability of human ?-globin mRNA in vivo. Nine Preliminary Studies provide compelling scientific rationale for the research aims detailed in this application. These studies use a novel in situ method to identify a previously unknown cis-determinant of p-globin mRNA stability, and demonstrate that this element is bound in trans by nucleolin, a multi- functional, structurally heterogeneous factor. Importantly, site-specific mutations that ablate nucleolin binding also destabilize the full-length ?-globin mRNA in situ in intact cultured cells. RNA-folding models predict a strong stem-loop structure within the ?-globin 3'UTR, encompassing the nucleolin-binding site in direct opposition to a functional binding site for ?CP, a factor that plays a critical role in regulating the stability of ?-globin mRNA. Based upon this data, we propose a model for ?-globin mRNA stability in which nucleolin remodels secondary structure within the p-globin 3'UTR to facilitate ?CP access to its functional binding site. The revised proposal contains new, corroborating studies demonstrating that nucleolin dramatically increases the affinity of ?CP for the native p-globin 3'UTR in vitro. New studies also demonstrate that nucleolin's ?-globin mRNA ligand-binding specificity is provided, at least in part, by its post-translational ribosylation. The current proposal extends the Preliminary Studies in three Aims that capitalize on the applicant's longstanding interest and relevant experience in the field of mRNA stability.
Aim I establishes a structural basis for the ?-globin mRNA ligand-binding specificity of nucleolin by identifying its participant RNA-binding domains, and by specifying the position and nature of relevant post-translational modifications.
Aim II validates the hypothesis that native secondary structure within the ?-globin 3'UTR prohibits ?CP binding to its functional binding site, and directly addresses the proposed role that nucleolin plays in remodeling this region.
Aim III defines other likely consequences of nucleolin-mediated 3'UTR remodeling, including effects on the processing and translational efficiency of nascent and mature ?-globin mRNAs, respectively. The Experimental Plan utilizes reagents and methods that have been validated in the applicant's laboratory, as well as creative approaches that will facilitate successful completion of the stated Aims. Results from the proposed experiments will afford unique insights into the molecular system that regulates ?-globin mRNA stability, providing a strong basis for the rational design of novel therapies for ? thalassemia and sickle cell disease that modulate this critical determinant of ?-globin gene expression.
| 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 |
| 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 |
| Abdulmalik, Osheiza; Safo, Martin K; Seeholzer, Steven H et al. (2010) Hb Baden: structural and functional characterization. Am J Hematol 85:848-52 |
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