Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Trophic factor deprivation (TFD) activates c-Jun N-terminal kinases (JNKs), culminating in coordinate AP1-dependent transactivation of the BH3-only BCL-2 proteins BIM(EL) and HRK, which in turn are critical for BAX-dependent cytochrome c release, caspase activation, and apoptosis. Here, we report that TFD caused not only induction but also phosphorylation of BIM(EL). Mitochondrially localized JNKs but not upstream activators, like mixed-lineage kinases (MLKs) or mitogen-activated protein kinase kinases (MKKs), specifically phosphorylated BIM(EL) at Ser65, potentiating its proapoptotic activity. Inhibition of the JNK pathway attenuated BIM(EL) expression, prevented BIM(EL) phosphorylation, and abrogated TFD-induced apoptosis. Conversely, activation of this pathway promoted BIM(EL) expression and phosphorylation, causing BIM- and BAX-dependent cell death. Thus, JNKs regulate the proapoptotic activity of BIM(EL) during TFD, both transcriptionally and posttranslationally.

Two new yeast genes, ASF1 (Anti‐Silencing Function) and ASF2, as well as a C‐terminal fragment of SIR3, were identified as genes that derepressed the silent mating type loci when overexpressed. ASF2 overexpression caused a greater derepression than did ASF1. ASF1 overexpression also weakened repression of genes near telomeres, but, interestingly, ASF2 had no effect on telomeric silencing. Sequences of these two genes revealed open reading frames of 279 and 525 amino acids for ASF1 and ASF2, respectively. The ASF1 protein was evolutionarily conserved. MCB motifs, sequences commonly present upstream of genes transcribed specifically in S phase, were found in front of both genes, and, indeed, both genes were transcribed specifically in the S phase of the cell cycle. While an asf2 mutant was viable and had no obvious phenotypes, an asf1 mutant grew poorly. Neither mutant exhibited derepression of the silent mating type loci. The asf1 mutant was sensitive to methyl methane sulfonate, slightly UV‐sensitive and somewhat deficient in minichromosome maintenance. It also lowered the restrictive temperature of a cdc13ts mutant. These phenotypes suggested a role for ASF1 in DNA repair and chromosome maintenance. The GenBank accession numbers for the ASF1 and ASF2 sequences are L07593 and L07649, respectively. © 1997 by John Wiley & Sons, Ltd.

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo. INTRODUCTIONTelomerase is a specialized reverse transcriptase that is essential for telomere maintenance. The telomerase ribonucleoprotein (RNP) uses an internal RNA template to synthesize telomeric repeat sequences onto chromosome ends. Deletion of the essential RNA component of telomerase leads to progressive telomere shortening, chromosome instability, and cell death in both yeast and mouse cells (Singer and Gottschling, 1994;McEachern and Blackburn, 1996;Blasco et al., 1997;Lee et al., 1998).The telomerase enzyme is made up of an essential core as well as several accessory proteins. The core telomerase enzyme consists of the RNA component (telomerase RNA [TR]) and the catalytic subunit (telomerase reverse transcriptase [TERT]). The structure of the RNA component is conserved (Romero and Blackburn, 1991;Lingner et al., 1994) in ciliates where the RNA is 150 -200 nucleotides in length (Greider and Blackburn, 1989). In mammalian cells the RNA component is significantly larger, 390 -450 nucleotides (Blasco et al., 1995;Feng et al., 1995), and the structure is highly conserved among vertebrates (Chen, Blasco, and Greider, unpublished results). The catalytic TERT component, first identified in the ciliate Euplotes (Lingner and Cech, 1996), has homologues in yeast (EST 2), human (hTERT), and mouse (mTERT) (Harrington et al., 1997b;Lingner et al., 1997b;Meyerson et al., 1997;Nakamura et al., 1997;Greenberg et al., 1998;Martin-Rivera et al., 1998). TERT contains sequence motifs similar to the conserved core of reverse transcriptases, and mutation of the conserved essential aspartate residues in the catalytic triad of reverse transcriptases eliminates telomerase activity (Lingner et al., 1997b;Weinrich et al., 1997). Minimal telomerase activity can be reconstituted in an in vitro transcription/translation extract using just the TERT and TR components Be...