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The yeast telomerase holoenzyme, which adds telomeric repeats at the chromosome ends, is composed of the TLC1 RNA and the associated proteins Est1, Est2 and Est3. To study the biogenesis of telomerase in endogenous conditions, we performed fluorescent in situ hybridization on the native TLC1 RNA. We found that the telomerase RNA colocalizes with telomeres in G1- to S-phase cells. Strains lacking any one of the Est proteins accumulate TLC1 RNA in their cytoplasm, indicating that a critical stage of telomerase biogenesis could take place outside of the nucleus. We were able to demonstrate that endogenous TLC1 RNA shuttles between the nucleus and the cytoplasm, in association with the Crm1p exportin and the nuclear importins Mtr10p–Kap122p. Furthermore, nuclear retention of the TLC1 RNA is impaired in the absence of yKu70p, Tel1p or the MRX complex, which recruit telomerase to telomeres. Altogether, our results reveal that the nucleo-cytoplasmic trafficking of the TLC1 RNA is an important step in telomere homeostasis, and link telomerase biogenesis to its recruitment to telomeres. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, Ash1p is a specific repressor of transcription that localizes exclusively to daughter cell nuclei through the asymmetric localization of ASH1 mRNA. This localization requires four cis-acting localization elements located in the ASH1 mRNA, five trans-acting factors, one of which is a myosin, and the actin cytoskeleton. The RNA-binding proteins that interact with these cis-elements remained to be identified. Starting with the 3′ most localization element of ASH1 mRNA in the three-hybrid assay, element E3, we isolated a clone corresponding to the C-terminus of She3p. We also found that She3p and She2p interact, and this interaction is essential for the binding of She3p with element E3 in vivo. Moreover, She2p was observed to bind the E3 RNA directly in vitro and each of the ASH1 cis-acting localization elements requires She2p for their localization function. By tethering a She3p-MS2 fusion protein to a reporter RNA containing MS2 binding sites, we observed that She2p is dispensable for She3p-MS2-dependent RNA localization. [ABSTRACT FROM AUTHOR]