Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Abstract

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.