Metabolic stability of 2′ 5′oligo (A) and activity of 2′ 5′oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells

Abstract

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.

Increased levels of (2'-5')oligo(A) polymerase activity in human lymphoblastoid cells treated with glucocorticoids.

“…1 for additional references.). Both enzymes require double-stranded RNA for activation and can be quantitated in cell extracts by recently developed assays (2,3). The first enzyme polymerizes ATP into a series of oligonucleotides characterized by (2'-5')phosphodiester bonds (4) and collectively designated (2'-5')oligo(A) or (2'-5')A.…”

Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An.

“…These agents are needed for the activation of an endonuclease activity,* but do not have to be present during the cleavage of RNA (6,7). The "nuclease activators" that mediate the activation are 2'-5' linked oligoadenylates [(2'-5')An] with 5'-terminal triphosphate or diphosphate (8)(9)(10)(11)(12)(13)(14)(15)(16). This series of compounds was originally discovered as an inhibitor of protein synthesis that is formed from ATP in extracts of various interferon-treated cells (and in lysates from rabbit reticulocytes not treated with interferon) in the presence of double-stranded RNA (17)(18)(19)(20).…”

“…This series of compounds was originally discovered as an inhibitor of protein synthesis that is formed from ATP in extracts of various interferon-treated cells (and in lysates from rabbit reticulocytes not treated with interferon) in the presence of double-stranded RNA (17)(18)(19)(20). The level of the enzyme catalyzing (2'-5')An synthesis is greatly increased in cells upon treatment with interferon (8,(21)(22)(23). The activation of the endonuclease by added (2'-5')An is transient in crude cell extracts.…”

“…It ceases, presumably, as a consequence of the degradation of (2'-5')An by enzymes (other than the endonuclease itself). Addition of further (2'-5')An can elicit renewed endonuclease activity (12,15,24). The activated endonuclease cleaves different single-stranded RNAs at widely different rates.…”

“…The enzyme was partially purified from mouse Ehrlich ascites tumor cells and L cells (8,11,12). In (25), the protein fraction tested for endonuclease activity (in [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Ml), and the specified amount of (2'-5')An preparation I, II, or III if so indicated. After incubation at 30°C for 1 hr, the reaction was terminated and the reaction mixture was analyzed by polyacrylamide gel electrophoresis and radioautography.…”