Schizosaccharomyces pombeCcq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase–telomere association

Abstract

The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415–507 of TER1. A ter1-Δ415–507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1–TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1–Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1Δ. We propose that the Est1–Ccq1 interaction is critical for telomerase recruitment, while the Est1–TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.

Replication of Telomeres and the Regulation of Telomerase

“…Telomerase recruitment to telomeres in S. pombe occurs through the interaction of the telomerase subunit Est1 with Ccq1, which is tethered to telomeres through the interaction with Tpz1, which in turn interacts with Pot1 and Poz1 (Miyoshi et al 2008;Tomita and Cooper 2008;Webb and Zakian 2012). Importantly, S. pombe Tel1 (ortholog of mammalian Atm) and Rad3 (ortholog of mammalian Atr) promote the direct interaction between Ccq1 and the Est1 subunit of telomerase (Moser et al 2011;Yamazaki et al 2012).…”

“…These subunits are not involved per se in catalysis, but they are important for telomerase assembly or the recruitment to telomeres. The S. cerevisiae Est1, which recruits telomerase to telomeres (discussed below), has a clear counterpart in S. pombe (Beernink et al 2003;Webb and Zakian 2012), but the orthologous relationship to human Est1-like proteins (referred to as Est1A/Smg6, Est1B/ Smg5, and Est1C/Smg7) is uncertain (Reichenbach et al 2003;Snowet al 2003). Est1A interacts as the budding and fission yeast Est1 proteins with telomerase, but a role in telomerase recruitment has not been shown (Redon et al 2007).…”

Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding

“…We previously showed that the terminus of the long TER1 arm binds Est1 (ever shorter telomeres protein 1) (16). The other region splits into two arms: One arm binds Sm and Lsm proteins (17), and the other is uncharacterized.…”

“…Southern and Northern blotting were performed as previously described (16). RNA immunoprecipitation and ChIP were performed as previously described (16). In vitro telomerase assays were performed as in ref.…”

“…ter1 mutants were constructed by using standard molecular genetic techniques. Southern and Northern blotting were performed as previously described (16). RNA immunoprecipitation and ChIP were performed as previously described (16).…”