Multiple yeast genes, including Paf1 complex genes, affect telomere length via telomerase RNA abundance.

Twofold reductions in telomerase RNA levels cause telomere shortening in both humans and the yeast Saccharomyces cerevisiae. To test whether multiple genes that affect telomere length act by modulating telomerase RNA abundance, we used real-time reverse transcription-PCR to screen S. cerevisiae deletion strains reported to maintain shorter or longer telomeres to determine the levels of their telomerase RNA (TLC1) abundance. Of 290 strains screened, 5 had increased TLC1 levels; 4 of these maintained longer telomeres. Twenty strains had decreased TLC1 levels; 18 of these are known to maintain shorter telomeres. Four strains with decreased TLC1 RNA levels contained deletions of subunits of Paf1C (polymerase II-associated factor complex). While Paf1C had been implicated in the transcription of both polyadenylated and nonpolyadenylated RNAs, Paf1C had not been associated previously with the noncoding telomerase RNA. In Paf1C mutant strains, TLC1 overexpression partially rescues telomere length and cell growth defects, suggesting that telomerase RNA is a critical direct or indirect Paf1C target. Other factors newly identified as affecting TLC1 RNA levels include cyclin-dependent kinase, the mediator complex, protein phosphatase 2A, and ribosomal proteins L13B and S16A. This report establishes that a subset of telomere length genes act by modulating telomerase RNA abundance.

Low abundance of telomerase in yeast: implications for telomerase haploinsufficiency.

Telomerase is an RNA-dependent reverse transcriptase that maintains telomeric DNA at a species-specific equilibrium length. To determine an upper limit for the number of telomerase molecules in a Saccharomyces cerevisiae cell, we have established real-time RT-PCR assays to quantify the noncoding telomerase RNA, TLC1. We find that the number of TLC1 molecules in a haploid yeast cell is approximately 29, less than the number of chromosome ends (64) in late S-phase. Wild-type diploid cells contain approximately 37 telomerase RNAs, while diploids heterozygous for a null tlc1 allele have half the wild-type amount, approximately 19 TLC1 molecules. For comparison, there are approximately 480 molecules of the U2 snRNA per haploid cell. We show that a biological consequence of this low level of telomerase is haploinsufficiency: A TLC1/tlc1Delta heterozygote maintains shorter telomeres. A dominant-negative telomerase RNA, with a deletion of the template for telomeric DNA synthesis, further demonstrates that yeast telomere length is sensitive to telomerase dosage. Sixfold overexpression of tlc1Deltatemplate establishes a new telomere length set point, approximately 160 bp shorter than wild type. Removing telomerase protein-interaction sites from the tlc1Deltatemplate RNA mitigates the dominant-negative effect, suggesting that the tlc1Deltatemplate RNA competes with wild-type TLC1 for a limited supply of telomerase proteins or for telomeres. Because yeast telomerase is tethered at chromosome ends, the finding that it may be outnumbered by its telomeric DNA substrates provides a new perspective for interpreting the results of telomere maintenance studies.

A fuzzy mitochondrial fusion apparatus comes into focus.

Membrane fusion is fundamental to eukaryotic life. Unlike the predominant intracellular fusion machineries that fuse compartments bounded by a single membrane, the mitochondrial fusion machinery must sequentially fuse the outer and inner mitochondrial membranes. These coordinated fusion events rely on a transmembrane GTPase that is known as fuzzy onions or Fzo. Recent studies have revealed that Fzo has an evolutionarily conserved role in mitochondrial fusion, and they take the first strides in determining the molecular nature of such a role.