Experiments have been outlined to approach the following problem: What are the molecular mechanisms regulating mRNA stability during differentiation of murine erythroleukemia cells? To address this problem we have developed a system in which: (1) cells reach the terminal stages of differentiation, become enucleated, and resemble normal reticulocytes; and (2) the entry of cells to the terminal differentiation program can be synchronized and occurs within 30 min. As differentiation proceeds in this system, nonglobin mRNA is dramatically destabilized but globin mRNA remains stable. We will test two hypotheses to account for this phenomenon: (1) the primary structure of mRNA changes during differentiation. This may occur via shifts in initiation or termination of transcription, via changes in processing machinery or via alterations in gene structure; and (2) genes are transcriptionally """"""""switched off"""""""" in a specific order. Those coding for the most stable mRNAs are """"""""switched off"""""""" first, while genes coding for less stable mRNA are """"""""switched off"""""""" later. To test these hypotheses we isolated mRNA from undifferentiated MEL cells and from """"""""superinduced"""""""" MEL cells. Using these preparations of mRNA, we constructed the corresponding cDNA libraries. The isolation of mRNA from induced cells and construction of the corresponding cDNA library presented a problem since even a slight contamination of induced cell population with uninduced cells resulted in a massive contamination of the mRNA and cDNA levels. To deal with this problem we developed several additional purification stages. Using our cDNA libraries we isolated a number of cDNA clones representing mRNA species which change their stability during differentiation process. These clones will be used to verify the hypotheses outlined above. We also will analyze changes in protein composition of specific ribonuclear protein (RNP) complexes during differentiation and try to correlate them with the changes in mRNA stability and in mRNA structure. The understanding of molecular mechanisms regulating differential mRNA stability may not only provide insights into the basis of abnormal growth and cancer, but will also enable us in the future to manipulate gene expression and perhaps correct some types of genetic defects such as beta thalassemia. (G)
| Volloch, V; Schweitzer, B; Rits, S (1987) Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells. J Cell Biol 105:137-43 |
| Volloch, V; Schweitzer, B; Rits, S (1987) Messenger RNA changes during differentiation of murine erythroleukemia cells. Exp Cell Res 173:38-48 |
| Volloch, V (1986) Cytoplasmic synthesis of globin RNA in differentiated murine erythroleukemia cells: possible involvement of RNA-dependent RNA polymerase. Proc Natl Acad Sci U S A 83:1208-12 |
