This is a new project submitted for consideration as a First Award, whose goal is to understand the molecular basis of the extraordinary stability of b-globin mRNA which allows it to accumulate to very high levels in developing erythroid cells. While progress has been made in understanding the basis of a-globin mRNA stability, none has yet investigated stability of the b-globin mRNA. The basic strategy used here will be similar to those applied to study of a-globin mRNA.
The first aim i s designed to define the cis determinants of stability within the b-globin mRNA. A variety of mutations will be introduced and the relative stability of normal and altered mRNA compared either in transfected MEL cells after transcriptional arrest, or in vivo in transgenic mice by comparing relative abundance in marrow versus circulating reticulocytes. Both assays utilize quantitative PCR methods.
The second aim will explore the trans-acting factors that bind with appropriate specificity to b-globin mRNA, presumably a protein or ribonucleoprotein complex that will protect it from degradation. In this aim MEL cell extracts will be incubated with b-globin mRNA transcripts under various conditions to optimize binding as assayed by native gel electrophoresis of RNase resistant complexes. The relevance of such complexes will be tested by correlating whether such binding correlates with stability using the battery of mRNA mutants produced in aim 1. To obtain more direct evidence the applicant proposes to affinity purify components of the RNP complex by using the b-globin mRNA probe, generate anti-complex antibodies, obtain microsequence results to facilitate cloning, etc. In vivo relevance is then proposed to be tested by assessing b-globin mRNA stability under conditions which disrupt the RNP complex. Several possible methods for disruption will be considered depending on the success in identifying and cloning b-complex components, including use of antisense RNA expression, overexpression of b-complex binding RNA sequences in a competition assay, or use of gene knockouts.
The third aim will explore the normal mechanism of b-globin mRNA degradation, and will attempt to distinguish between decapping, poly A tail shortening, or endonucleolytic cleavage as initiators of degradation. These experiments will utilize transgenic mice expressing high levels of normal or mutant human b-globin mRNA.
| Yu, J; Russell, J E (2001) Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA. Mol Cell Biol 21:5879-88 |
