After interferon‐gamma (IFN‐gamma) treatment of cells the appearance of tyrosine phosphorylated Stat1 in the nucleus was maximal within 20–30 min, remained for 2–2.5 h and activated molecules disappeared by 4 h. In the absence of continued signaling from the receptor (imposed by staurosporine treatment) previously activated Stat1 disappeared completely within 60 min, implying continuous generation and removal of active molecules during extended IFN‐gamma treatment. Proteasome inhibitors prolonged the time of activation of Stat1 by prolonging signaling from the receptor but not by blocking removal of already activated Stat1 molecules. By analyzing with 35S labeling the distribution of total Stat1 and activated Stat1, we concluded that the Stat1 molecules promptly cycle into the nucleus as tyrosine phosphorylated molecules and later return quantitatively to the cytoplasm as non‐phosphorylated molecules. Therefore, the removal of the activated Stat1 molecules from the nucleus appears not to be proteolytic but must depend on a protein tyrosine phosphatase(s).

Poly(A)-containing HeLa cell mRNA prepared from cells labeled with [3Plphosphate was found to contain a variety of methylated, blocked 5'-terminal structures of two general types: m7GpppNm-Np and m7GpppNm-Nm-Np. In addition, about one-third of the [3Hlmethyl label was present in the N6-methyladenosine; this labeled nucleoside was not found in the 3'-terminal one-third of the mRNA chain and thus may also be in the 5' portion of the mRNA.The 5' end of a variety of viral mRNA molecules consists of an unusual type of methylated oligonucleotide with the general structure m7G(5')ppp(5')Nm-Np (1-6). Because the addition through pyrophosphate linkage of the terminal m7G renders the terminal dinucleotide resistant to digestion by the usual ribonucleases, the structure has been called a "cap" (7). Perry and Kelley (8) have shown that mammalian cell mRNA contains methyl groups, and, with the availability of methods used to characterize the cap structures in viral mRNAs, we have examined HeLa cell mRNA for the presence of caps. As pointed out by Rottman et al. (7), information about various steps of methylation of potential mRNA precursors by the cell should aid in understanding eukaryotic mRNA formation. MATERIALS AND METHODSRadioactive Labeling. Growth of suspension cultures of HeLa cells was in Eagle's medium. Cells were labeled with 32p for 4 hr in phosphate-free medium as described (9). Labeling with [methyl-3H]

Interferon a induction of transcription operates through interferon-stimulated-gene factor 3 (ISGF), a transcription factor two components ofwhich are members of the newly characterized Stat family of transcription factors.Interferon a induces tyrosine phosphorylation of Statl and Stat2 proteins that associate and, together with a 48-kDa protein, form ISGF3. Evidence is presented that a heterodimer of Statl and Stat2 is present in ISGF3 and that Statl and the 48-kDa protein make precise contact, while Stat2 makes general contact, with the interferon-stimulated response element, the binding site of the ISGF3.Cytokine attachment to cell surface receptors triggers gene activation in the cell nucleus (1). Many of these extracellular polypeptides cause their intracellular changes through the Jak-Stat pathway (2). The Jak proteins are protein-tyrosine kinases associated with cell surface receptors that are activated by receptor occupation. The Stat proteins serve the dual function of signal transduction and activation of transcription. The first polypeptide ligand recognized to use this pathway was interferon a (IFN-a), which leads to activation of a nuclear DNA-binding complex called interferon-stimulated-gene factor 3 (ISGF3) (3, 4), which upon purification proved to contain four protein species, 113, 91, 84, and 48 kDa in size (5). The first three of these were the proteins that yielded sequence information establishing the sequence similarity in the Stat family (6, 7); it was also demonstrated that the 113-, 91-, and 84-kDa proteins (renamed Stat2, Statla, and Statlf3, respectively) were activated by phosphorylation on a single similarly located tyrosine (8-10). (The 91-and 84-kDa proteins differ only in a 38-aa carboxyl-terminal extension in the 91-kDa protein.) The 48-kDa protein,

Each poly(A) containing cytoplasmic AD-2 MRNA contains at its 5' terminus the general structure m7 GpppN1 pN2p or m7 GpppN1mpN2mpNp as well as an average of 4 m6A and 0.5-1 m5C residues per molecule. Almost all of the N1m residues are adenine derivatives including Am, m6Am and probably m26,6Am. The N2m is mostly Cm but small amounts of the other three methylated bases are also present. All the methylated constitutents of mRNA are distant from the 3' terminal poly(A). The amount of m6A appears to be greater in larger mRNA than in smaller mRNA. Nuclear Ad-2 specific RNA also contains caps, m6A, and m5C with about twice as much m6A relative to caps as cytoplasmic mRNA. The similarity of Ad-2 nuclear and mRNA to HeLa hnRNA and mRNA suggests that adenovirus mRNA production is a good model for eukaryotic mRNA production.

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