Molecular cloning and sequence of partial cDNA for interferon-induced (2'-5')oligo(A) synthetase mRNA from human cells.

Abstract

By using a translation assay in oocytes, a 17S RNA fraction coding for the interferon-induced (2'-5')oligo(A) synthetase was purified from human cells. A cDNA library was prepared by cloning in Escherichia coli plasmid pBR322 and screened by positive hybridization-translation in oocytes. A cDNA clone corresponding to the (2'-5')oligo(A) synthetase mRNA was identified.In SV80 cells, this E cDNA recognizes three RNAs of 1.65, 1.85, and 3.6 kilobases, which are present only after interferon treatment of the cells. In Namalva cells, mainly one RNA of 1.8 kilobases is seen.All interferons (IFNs) induce in cells the synthesis of a certain number of proteins and their mRNAs (1-3). These induced proteins include translation regulatory enzymes (1-3), surface antigens (4-6), and unidentified proteins (7-9) and are probably involved in the complex antiviral, antimitogenic, and immunoregulatory effects of IFNs. Among the enzymes induced by IFNs, the (2'-5')oligo(A) synthetase has been best studied. This enzyme binds double-stranded (ds) RNA and polymerizes ATP into ppp(A2'p)nA dimers, trimers, and longer oligomers (10), which activate the latent RNase F (11-13), thereby inhibiting protein synthesis. The (2'-5')oligo(A) synthetase increases 10-to 10,000-fold in various vertebrate tissues and cell lines, exposed to IFN-a, -P, or -y (1-3, 14). Induction of the (2'-5')oligo(A) synthetase and activation of RNase F seem to be a central mechanism, although not unique (15), in IFN's antiviral action (1-3, 16) and in the decrease of cell proliferation (17,18 In SV80 cells, the E cDNA hybridizes to three cytoplasmic RNA species of 1.65, 1.85, and 3.6 kb, whereas in Namalva cells, the predominant RNA is 1.8 kb.MATERIALS AND METHODS Cell Cultures and IFNs. Human SV80 cells were grown to confluent monolayers of 5 x 107 cells and lymphoblastoid Namalva cells were grown in suspension to 1.5 x 106 cells per ml as reported earlier (6). Human IFN-(31 prepared (6, 25) and partially purified (26) to 107 units/mg of protein was used to treat cells at 200 units of IFN-j31 per ml for 12 hr, unless otherwise indicated. IFN-/31 (>2 x 108 units/mg) purified by monoclonal antibody affinity chromatography and IFN-a were obtained as described (27,28).mRNA Isolation and cDNA Cloning. Total cell RNA was prepared as reported earlier (6) with LiCl/urea (29) and purified on oligo(dT)-cellulose (yield, 0.4 mg per 109 SV80 cells). The 17S RNA fraction from IFN-treated cells was isolated (unpublished data) in a preparative gel electrophoresis apparatus in 1.5% agarose/6 M urea/25 mM sodium citrate, pH 3.5 (30), and repurified on oligo(dT)-cellulose. ds cDNA was made from 2 ,g of 17S RNA as detailed (9, 31). Both strands were made with avian myeloblastosis virus reverse transcriptase (J. Beard) by priming with (oligo)dT, the first strand being tailed with dATP prior to second-strand synthesis (32). Blunt ends were formed on the ds cDNA (9) and the largest fractions were tailed with dCTP and hybridized to dGTP-tailed, Pst I-cut pBR322 DNA. E. coli MM294...

Diverse functions of RNase L and implications in pathology

“…The role played by RNase L in IFN-induced antiviral activity has clearly been demonstrated by transfection of 2-5A or stabilized 2-5A analogues in intact cells [49][50][51][52][53][54]. The cloning of 2-5A-synthetase [55,56] and RNase L [1] allowed the definitive demonstration of the antiviral activity of RNase L. The constitutive expression of the 40 kDa 2-5A-synthetase confers resistance to EMCV and mengovirus [57,58] and over expression of the 69 kDa form of 2-5A-synthetase protein in human cells inhibits the replication of EMCV [59]. Similarly, the antiviral activity of IFN against EMCV was inhibited by the expression of a dominant negative mutant of RNase L [28] or treatment with a 2-5A antagonist [49,53,54].…”

A scientific journey through the 2-5A/RNase L system

“…When stimulated by dsRNA, the functional OAS proteins produce a series of short 5′-phosphorylated, 2′,5′-linked oligoadenylates collectively referred to as 2-5A [p x 5′A(2′p5′A) n ; x = 1-3; n≥2] from ATP [9]. The first molecular clone for an OAS was obtained by Michel Revel's lab [10]. Biochemical characterization of OAS proteins by Ara Hovanessian's lab [11][12][13][14] and Ganes Sen's lab [15][16][17][18][19] and a crystal structure by Rune Hartmann and Vivien Yee (in collaboration with Ganes Sen and Just Justesen) [20] have led to functional and structural insight into the OAS family of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.…”

Regulation of synthesis and turnover of an interferon-inducible mRNA.

“…However, it is known that the expression of several genes is enhanced in IFN-treated cells. The products of these IFN-inducible genes presumably carry out various actions of IFN. Recently, partial cDNA clones of several human and murine IFN-inducible mRNAs have been isolated and used for studying the nature of regulation of synthesis and turnover of these mRNAs in IFN-treated cells (1,6,8,10,13,15,16). Enhanced transcription of some IFN-inducible mRNAs can be detected within minutes of IFN coming in contact with cells (3,6,8).…”

“…The steady-state cytoplasmic level of a particular IFN-inducible mRNA is dependent not only on its rate of transcription but also on its rate of turnover. The turnover rate is again different for different IFN-inducible mRNAs, and the turnover rate for a particular mRNA may vary with time elapsed after IFN treatment has begun (1,6,8,13,15). All IFN-inducible mRNAs studied so far seem to be the products of primary response to IFN, in the sense that they are induced by IFN in the presence of inhibitors of protein synthesis (1,6,8,10,13,15).…”