Specific RNA-cleaving activities from HeLa cells.

Abstract

It is now evident that in primary transcripts of most prokaryotic and eukaryotic RNAs the regions destined to occur in the mature molecules are interspersed among regions that are removed during the maturation process (1-5). The role of RNA processing enzymes is the correct removal of these "extra" regions, leading to the formation of functional mature RNAs. Because of the availability of numerous well-defined prokaryotic RNA precursors, the enzymatic RNA processing reactions that occur in prokaryotes have been the most extensively studied (6-10). From these studies the conclusion has emerged that such enzymes retain the ability to cleave their natural substrates properly in vitro, even after extensive purification of both the enzyme and the RNA precursors. Considerably less information concerning the enzymes involved in specific cleavage of eukaryotic RNA precursors (11, 12) is available.We have carried out gentle fractionation of HeLa cells to minimize release of random nucleases, and we have assayed nuclear and cytoplasmic fractions for their ability to cleave a wide spectrum of well-defined prokaryotic and eukaryotic RNA molecules. Four specific RNA-cleaving activities were detected: cleavage of heterogeneous nuclear RNA (hnRNA) in a characteristic fashion, specific cleavage of 45S rRNA precursor, cleavage of RNA-DNA hybrids (RNase H), and cleavage of a tRNA precursor (RNase P). Only the latter two activities have been described previously in mammalian cells. MATERIALS AND METHODSCell Growth and Subcellular Fractionation. HeLa (human) cells (strain S3) from the collection of Igor Tamm, The Rockefeller University, were maintained in suspension culture [Joklik's modified Eagle's medium (GIBCO) containing 5% fetal calf serum (GIBCO)]. Cells from 1-3 liters of culture at 5-6 X 105 cells per ml were harvested and fractionated according to Bothwell and Altman's modifications (13) of methods published by Penman (14) and Amaldi and Attardi (15). Worthington DNase DPFF was used without further treatment in the preparation of nuclear lysates. Ammonium sulfate precipitation was performed by adding neutralized saturated ammonium sulfate (40C) to obtain the desired final concentration, with constant stirring, incubating at 40C for 20 min, and centrifuging at 20,000 X g for 20 min. Precipitates were resuspended in 10 mM Tris-HCl, pH 7.5/20 mM NaCI/2 mM MgCl2/1 mM dithiothreitol, and dialyzed for 2 hr at 40C against this buffer.Labeled Enzyme Assays. Protein concentrations were determined by measuring the spectral shift caused by Coomassie blueprotein interaction as described by Bradford (21). Comparable proportions of subcellular fractions were assayed for their ability to cleave RNA substrates by incubation in 10 ,l of a standard buffer containing 30 mM Tris-HCI at pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 mM dithiothreitol for 30 min at 37°C. Reactions were terminated by adding 3 ,ul of 20% sucrose/1% sodium dodecyl sulfate/0.2% bromphenol blue/0.2 M EDTA. The extent of substrate cleavage was determined by ...

Viral tRNA Mimicry from a Biocommunicative Perspective

“…The results from the competition are very similar to those obtained by Dr. Engelke’s group using RNase P and yeast mRNAs Figures 2B,C in Marvin et al (2011) , although that study did not provide data on the competition with the pre-tRNA. However, direct examination of the products from the mRNA population after incubation with human RNase P did not reflect processing, which was also in line with studies published in the 1970s that had tested the cleavage of hnRNA using RNase P-enriched extracts ( Ferrari et al, 1980 ; Kole and Altman, 1981 ), and did not observe processing. When instead of examining the mRNA population, human or yeast mRNA species were tested separately ( Marvin et al, 2011 ; Díaz-Toledano and Gómez, 2015 ), it was seen that these mRNAs were sensitive to being processed by RNase P in specific positions, but there was a very low cutting effectiveness.…”

Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA

“…When such regions were found, they provided important information regarding the processing or function of the RNAs carrying them [ 64 – 67 ]. As this data began to emerge, it was shown that internally labelled hnRNA contained little or no target sites for RNase P from E. coli and HeLa cellular fractions [ 56 , 68 ]. As these findings suggested that RNase P did not participate in any of the steps in which mature mRNAs were cleaved from longer primary transcripts, there seemed to be no role for RNase P in mature mRNA turnover/metabolism [ 56 , 68 ].…”

“…As this data began to emerge, it was shown that internally labelled hnRNA contained little or no target sites for RNase P from E. coli and HeLa cellular fractions [ 56 , 68 ]. As these findings suggested that RNase P did not participate in any of the steps in which mature mRNAs were cleaved from longer primary transcripts, there seemed to be no role for RNase P in mature mRNA turnover/metabolism [ 56 , 68 ]. Probably because of this fact, the specificity and proportion, if any, of RNase P-sensitive sites within eukaryotic mRNA species remains unknown.…”

Structure and distribution of Alu family sequences or their analogs within heterogeneous nuclear RNA of HeLa, KB, and L cells.

“…The possibility that AfRNA is part of an RNA processing signal (whether in single-or double-stranded form) remains attractive since all dAfRNA (and nearly all AfRNA) is somehow removed from mRNAs before their transport from nucleus to cytoplasm is completed (15,29). A survey of HeLa cell nuclear and cytoplasmic extracts in our laboratory has not yet revealed an activity which (like E. coli RNase III) cleaves within dAfRNA regions (10). On the other hand, Nilsen et al (24) have recently reported that the (2',5')oligoadenylate-dependent endonuclease, RNase L, of HeLa cells can cleave HeLa cell hnRNA near the characteristic dAfRNA regions.…”