2Ј,5Ј-Oligoadenylate-dependent RNase L functions in the interferon-inducible, RNA decay pathway known as the 2-5A system. To determine the physiological roles of the 2-5A system, mice were generated with a targeted disruption of the RNase L gene. The antiviral effect of interferon α was impaired in RNase L -/-mice providing the first evidence that the 2-5A system functions as an antiviral pathway in animals. In addition, remarkably enlarged thymuses in the RNase L -/-mice resulted from a suppression of apoptosis. There was a 2-fold decrease in apoptosis in vivo in the thymuses and spleens of RNase L -/-mice. Furthermore, apoptosis was substantially suppressed in RNase L -/-thymocytes and fibroblasts treated with different apoptotic agents. These results suggest that both interferon action and apoptosis can be controlled at the level of RNA stability by RNase L. Another implication is that the 2-5A system is likely to contribute to the antiviral activity of interferon by inducing apoptosis of infected cells.

RNase L, a key enzyme in the anti-viral activity of interferons, requires activation by 2',5'-linked oligoadenylates (2-5A) to cleave viral and cellular single-stranded RNA. Here we demonstrate that 2-5A causes formation of stable dimers of RNase L in intact human cells as measured with a mammalian two-hybrid system. Hybrid proteins consisting of the GAL4 DNA binding domain fused to RNase L and the VP16 transactivation domain fused to RNase L were able to associate and drive transcription of a reporter gene, but only after cells were transfected with 2-5A. Several functional forms of 2-5A, such as p3A2'p5'A2'p5'A, were capable of activating transcription in human HeLa cells. In contrast, p3A2'p5'A, which can neither activate nor dimerize RNase L, did not induce gene expression. Evidence for the involvement of the C-terminal region of RNase L in dimerization was obtained by expressing truncated forms of RNase L. These findings describe a convenient, high-throughput screening method for RNase L activators which could lead to the discovery of novel anti-viral and anti-cancer agents.

We have characterized the 5'-flanking region of the alpha-subunit gene of the human pyruvate dehydrogenase (E1). DNase I footprinting with rat liver nuclear extracts identified 7 major protein-binding domains termed P1 through P7 in a 796 base pair DNA fragment (base pairs -763 to +33). P1 through P4 are clustered in the -221/+33 region. These protein-binding domains contain several known consensus sequences such as a TATA box, CAAT box, Sp1, and CRE, which all have previously been implicated in the constitutive transcription of several genes. Oligonucleotide competition studies indicate that oligonucleotides specific for CTF/NF-1 and Sp1 displaced the nuclear proteins bound to the CAAT box (within P3) and an Sp1 site (within P4), respectively. Several other well-characterized and purified transactivators (c-Fos, c-Jun, C/EBP, AP-2, and Sp1) have been shown to bind to the -221/+33 region. Other elements located upstream of the -221/+33 region, which includes nuclease protection domains P5-P7, are required for enhanced promoter activity of the 796 bp sequence. Promoter activity was measured by transient expression of a chloramphenicol acetyltransferase gene ligated to deletion fragments of the 5'-flanking region. Crucial element(s) for promoter activity and complex DNA-nuclear protein interactions were confined within a region spanning -221/+33. This region also retained more than 75% of the promoter activity of the 796 bp sequence. Additionally, this promoter region shows characteristics of both facultative and housekeeping gene promoters, suggesting complex transcriptional regulation.

A genomic clone (19 kb) harboring the intron-exon sequences and the promoter-regulatory region of the E1 beta gene of human pyruvate dehydrogenase complex was isolated by screening a placental genomic library. The nucleotide sequence of the promoter region (1245 bp) showed 18 differences (including mismatches, insertions, and deletions) as compared to the published sequence [Koike et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5594-5597]. The E1 beta promoter lacked a TATA box homology but contained initiator sequences (two) and Sp1 sites (three) which are frequently found in TATA-less promoters. The DNase I footprinting pattern of the promoter region with crude rat liver nuclear extracts showed at least seven regions of protein binding and nuclease protection (P1-P7). The DNase I protected regions contained consensus nucleotide sequences recognized by GATA-1, Sp1, IgNF-A, Lva, bicoid Q9, NF-kB, HNF-5, H4TF-1, WAP5, and ADH transription factors. Transient expression of chloramphenicol acetyltransferase (CAT) suggested the possible presence of negative elements located within the sequence from -2316 to -930, whereas deletion constructs containing -929 to +32 and -98 to +32 DNA sequences showed approximately 7- and 20-fold increases in CAT activity over the basal CAT activity. Additional studies indicated the presence of an orientation-dependent cis element (or elements) within the region from -282 to -397 that acts as an enhancer or a repressor upon a heterologous thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

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