Specific Inhibition of Pre-Ribosomal RNA Processing in Extracts from the Lymphosarcoma Cells Treated with 5-Fluorouracil1

from the different laboratories may, in part, be due to the use of the different techniques in the experiments. It is evident from these studies that the precise mechanism by which 5-FUra inhibits cellular prolif eration and exerts its cytotoxic effects has not been fully elucidated. Early electron microscopic studies showed alterations in the nucleo lar size after 5-FUra treatment (9). Using antibodies specific for nucleolar structures, Greenhalgh and Parish (6) observed increase in nucleolar size following treatment with the drug, which might arise from its deleterious effects on rRNA processing and the resultant accumulation of rRNA precursors. To dissect the action of 5-FUra on rDNA transcription by RNA polymerase I and on pre-rRNA process ing, it is important to study its action in cell-free extracts that can correctly transcribe rDNA and process the transcript. The in vitro system can also facilitate identification and subsequent characteriza tion of the trans-acting factors involved in these reactions, which may be modified by 5-FUra treatment. For several years, our laboratory has been studying the mechanism and regulation of rDNA transcription (10—13).The availability of an efficient in vitro system from the animal cells facilitated these studies. This system can now be used to study the effect of 5-FUra or other related drugs on rDNA transcription without interference from other factors. rRNA in mammalian cells is initially synthesized as a large precur sor (14—16)which is subsequently processed into mature 28S, 18S, and 5.85 RNAS. The first step in the processing of pre-rRNA is the endonucleolytic cleavage near the 5 â€ẽnd of the external transcribed spacer, approximately 4 kilobases upstream from the 185 start site. The 5' cleaved product is rapidly degraded and the 3' end product is further processed by both endoand exonucleolytic cleavages to yield the mature RNM. The processing at the primary site occurs at a pair of cleavage sites located approximately 5 nt apart (17). Recently, we have turned our attention to elucidation of the trans acting factors that are involved in the pre-rRNA processing. As a first step toward achieving this goal, we have developed an in vitro pro cessing system from the mouse lymphosarcoma cells, which can ac curately process pre-rRNA at the primary processing site. Using this system, we examined the effect of 5-FUTP on rDNA transcription and processing. This study has shown that incorporation of 5-FUTP into RNA did not affect rDNA transcription or rRNA processing whereas pretreatment of cells with 5-FUra resulted in either inactivation or decreased synthesis of a trans-acting factor(s) that is directly involved in pre-rRNA processing. Abstract