The 2-5 oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.The 2Ј-5Ј oligoadenylate (2-5A)/RNase L system is an interferon (IFN)-inducible RNA degradation pathway which is responsible for many of the antiviral and antiproliferative effects of IFNs (37, 41).The 2-5A pathway is composed of at least three types of enzymatic activities: 2-5A-synthetase, 2-5A-degrading enzymes, and RNase L. 2-5A, an oligoadenylate with 2Ј-5Ј phosphodiester bonds, activates RNase L (53), a latent endoribonuclease. Upon activation, RNase L cleaves mRNAs 3Ј of UpNp sequences, thus leading to the inhibition of protein synthesis (14,21).The activity of RNase L was originally thought to be modulated solely by the concentration of the 2-5A activator (11,21). Moreover, we have previously established that RNase L activity can also be regulated by RLI (RNase L inhibitor), a protein inhibitor (5). Overexpression of the RLI cDNA in HeLa cells results in the inhibition of the IFN-activated 2-5A pathway. RLI is induced by viruses such as encephalomyocarditis virus (EMCV) and human immunodeficiency virus (HIV), causing an inhibition of the 2-5A/RNase L system (27, 28). The role of the 2-5A/RNase L pathway in the selective reduction of viral mRNA during EMCV and HIV infection has been demonstrated elsewhere (16,25,27).Variations in intracellular 2-5A and 2-5A-synthetase levels have been observed during cell growth and differentiation even in the absence of exogenous IFN treatment. Indeed, expression of IFN-inducible proteins, such as 2-5A-synthetase, doublestanded RNA-activated protein kinase (PKR), and p202 (a member of the "200 family" of murine proteins) h...

The 2-5A/RNase L system is considered as a central pathway of interferon (IFN) action and could possibly play a more general physiological role as for instance in the regulation of RNA stability in mammalian cells. We describe here the expression cloning and initial characterization of RLI (for RNase L inhibitor), a new type of endoribonuclease inhibitor. RLI cDNA codes for a 68-kDa polypeptide whose expression is not regulated by IFN. Its expression in reticulocyte extracts antagonizes the 2-5A binding ability and the nuclease activity of endogenous RNase L or the cloned 2DR polypeptide. The inhibition requires the association of RLI with the nuclease and is dependent on the ratio between the two proteins. Likewise RLI is coimmunoprecipitated with the RNase L complex by a nuclease-specific antibody. RLI does not lead to 2-5A degradation or to irreversible modification of RNase L. The overexpression of RLI in stably transfected HeLa cells inhibits the antiviral activity of IFN on encephalomyocarditis virus but not on vesicular stomatitis virus. RLI therefore appears as the first described and potentially important mediator of the 2-5A/RNase L pathway.

The antiviral and antiproliferative effects of interferons are mediated in part by the 2'-5' oligoadenylate-RNase L RNA decay pathway. RNase L is an endoribonuclease that requires 2'-5' oligoadenylates to cleave single-stranded RNA. In this report we present evidence demonstrating a role for RNase L in translation. We identify and characterize the human translation termination factor eRF3/GSPT1 as an interacting partner of RNase L. We show that interaction of eRF3 with RNase L leads to both increased translation readthrough efficiency at premature termination codons and increased +1 frameshift efficiency at the antizyme +1 frameshift site. On the basis of our results, we present a model describing how RNase L is involved in regulating gene expression by modulating the translation termination process.