Short capped hnRNA precursor chains in HeLa cells: continued synthesis in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

Abstract

The labeling of m7GpppN1mpN2p caps with L-[methyl-3H]methionine on short (100-500 nucleotides) heterogeneous nuclear RNA (hnRNA) chains of HeLa cells is increased 2-3 times but the labeling of caps on longer (greater than 2000 nucleotides) hnRNA chains is decreased by approximately 80% by treatment of the HeLa cells with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The experimental conditions were as follows: HeLa cells were treated with 75 muM DRB for 40 min before labeling and also during the 30-min pulse of L-[methyl-3H]methionine; actinomycin D (0.05 microgram/mL) was used to suppress ribosomal RNA synthesis. Control cells received no DRB. The RNA was separated in Me2SO gradients to ensure no aggregation. Labeling of cells with [3H]uridine for 10 min and separation of RNA by these techniques reconfirmed the findings [Tamm, I., Hand, R., & Caliguiri, L. A. (1976) J. Cell Biol. 69, 229-240; Sehgal, P. B., Darnell, J. E., Jr., & Tamm, I. (1976) Cell 9, 473-480] that 70-80% of the synthesis of hnRNA (GREATER THAN 1000 NUCLEOTIDES) IS SENSITIVE TO INHIBITIOn by DRB but that 20-30% is resistant. This analysis of the methyl-labeled caps provides evidence that DRB causes early termination of a large fraction (approximately 70-80%) of hnRNA precursor chains. In contrast to the finding of continued synthesis and accumulation of short m7GpppN1mpN2p-capped chains in the presence of DRB, the synthesis of m2,2,7GpppN1mpN2mp-capped small nuclear RNAs was inhibited by approximately 70% by DRB.