Christopher J. Webb and Virginia A. Zakian

GENETICS

Significance

We demonstrate that the fission yeast telomerase RNA has a stem terminus element (STE) that it is essential for telomerase action in vivo and in vitro. Using a partial loss-of-function STE allele, we show that the STE is required for wild-type telomeric sequence. This is the first example, to our knowledge, of a sequence that is not part of the telomerase RNA core region that affects the sequence of telomeric DNA. Because mutating the STE has the same phenotypes as mutating the template boundary element (TBE), the STE promotes TBE function. The association of two sequence-specific telomere binding proteins is impaired in the STE mutant. Thus, the STE is critical to assemble the normal sequence and chromatin structure of fission yeast telomeres.

Abstract

The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

See: http://www.pnas.org/content/112/36/11312.abstract

PNAS September 8, 2015; vol.112, no. 36: 11312–11317

Fig. S2.

TER1 overexpression does not cause short telomeres. (A) Southern blots. Genomic DNA was extracted from cells harboring pSP1 and ter1/pSP1 after seven successive streaks. DNA was digested with ApaI and hybridized to a telomeric probe. Molecular weight markers are in kb. (B) Northern blot. Total RNA was extracted from wild-type cells containing either pSP1 vector or pSP1 carrying ter1 expressed from its own promoter [ter1/pSP1 (6)]. Northern blotting was performed as described in Fig. 1D. Relative levels of TER1 normalized to the U2 signal are indicated below the blot. (C) TER1 and TER1-STE_loop RNAs localize to the nucleus. Northern blot of RNAs isolated from cytoplasmic and nuclear fractions. TER1 was identified by hybridization as in Fig. 1D. Effective fractionation was demonstrated by localization of the tRNA_his to the cytoplasmic fraction. (D) Nuclear and cytoplasmic RNAs analyzed in C were used as substrates for quantitative RT-PCR analysis. RNAs detected in each fraction are expressed as percent of the nuclear plus cytoplasmic RNAs. Error bars are SDs. The amounts of total fractionated RNA were determined by Nanodrop spectrophotometer. (E) FISH. Cells expressing TER1 or TER1-STE_loop were fixed and hybridized with TER1 (Quasar 670) fluorescence probes (Biosearch Technologies). Nuclear DNA is indicated by DAPI staining.