Messenger and Heterogeneous Nuclear RNA in HeLa Cells: Differential Inhibition by Cordycepin

Abstract

Abstract. suppresses the labeling of messenger RNA in HeLa cells. The drug has no effect on either the labeling of nuclear heterogeneous RNA or on the transport of messenger RNA into the cytoplasm. The results suggest that messenger RNA and nuclear heterogeneous RNA are synthesized separately, and that the transcription of messenger RNA is inhibited by the drug.The nuclei of metazoan cells contain giant heterogeneous RNA. This species constitutes a major fraction of the RNA labeled during a pulse and turns over rapidly.5 Its function remains to be defined.Some properties of the heterogeneous nuclear RNA (HnRNA) resemble those expected of cytoplasmic messenger RNA (mRNA). It is heterogeneous in size, its G + C content resembles that of cellular DNA, and it apparently hybridizes to a considerable fraction of the genome (J. E. Darnell, personal communication). However, a closer examination shows that HnRNA is actually quite different from mRNA found on polyribosomes. HnRNA ranges in sedimentation values from 10 S to greater than 200 S and is much larger than the mRNA found on cytoplasmic polyribosomes (8 S-30 S).6 While mRNA is found on polyribosomes in the cytoplasm, HnRNA is confined to the nucleus where no proteinsynthesizing structures have been found, at least after cell fractionation. The rate of labeling of HnRNA is much greater than that of cytoplasmic mRNA.One hypothesis that attempts to reconcile the disparate properties of these RNA species postulates that HnRNA is a precursor to cytoplasmic mRNA and that a small region of the large molecule is cleaved and exported to the cytoplasm.7 However, the kinetics of labeling of HnRNA and mRNA are very different and do not seem to support a simple precursor-product relation6 although conclusions drawn from such measurements are far from compelling.The experiments reported here also suggest that mRNA is not derived by cleavage from HnRNA, but rather is synthesized separately. Cordycepin, 3'-deoxyadenosine, a drug that appears to act primarily by interfering with transcription, selectively suppresses the labeling of cytoplasmic mRNA while it does not affect that of HnRNA.Materials and Methods. Cell culture: HeLa cells were grown in suspension culture in Eagle's medium as described.8 The cell density was 4 X 105 cells/ml during growth and 2 X 106 cells/ml during labeling.1878

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

“…Because cordycepin interferes with polyadenylation, it was concluded that the probe must have been hybridized in the untreated cells. However, cordycepin has not been used in previous studies on mammalian cell nuclear RNA metabolism for more than 1-3 h (Siev et al, 1969;Penman et al, 1970;Darnell et al, 1971;Mendecki et al, 1972) because of severe toxicity effects. Moreover, it has been clearly established that cordycepin inhibits the production of cytoplasmic mRNA in HeLa cells within 25-40 min (Penman et al, 1970).…”

“…However, cordycepin has not been used in previous studies on mammalian cell nuclear RNA metabolism for more than 1-3 h (Siev et al, 1969;Penman et al, 1970;Darnell et al, 1971;Mendecki et al, 1972) because of severe toxicity effects. Moreover, it has been clearly established that cordycepin inhibits the production of cytoplasmic mRNA in HeLa cells within 25-40 min (Penman et al, 1970). The mRNA population of HeLa cells consists of at least two kinetic components; 30 -40% has an average half-life of 2-7 h, and the rest has an average halflife of 24 h (Singer and Penman, 1973;Puckett and Darnell, 1977).…”

Inhibition of polyadenylation reduces inflammatory gene induction

“…Because of its structure, cordycepin could potentially interfere with any process requiring adenosine or ATP. In particular, cordycepin triphosphate was proposed to be a chain terminator for transcription, but it was reported early that total nuclear mRNA precursors are less affected than cytoplasmic mRNA (Penman et al 1970). In contrast, it has been demonstrated to be an efficient chain terminator for mRNA polyadenylation by poly(A) polymerases (Rose et al 1977).…”

Comparison of growth‐inhibiting activities of Cordyceps militaris and Paecilomyces japonica cultured on Bombyx mori pupae towards human gastrointestinal bacteria

“…8 Submerged cultivated C. militaris is found to secrete cordycepin into the environment. 20 Cordycepin is found to possess antitumor activity, 21 inhibition of nuclear RNA synthesis in nerve and glial cells, 22 and antiviral activity towards HIV-1 in vitro, 23 as well as antiprotozoal effect 24 and insecticidal activity against Plutella xylostella (L.). 25 Structure-growth inhibition relationships in intestinal organisms have been studied.…”