Biogenesis of yeast telomerase depends on the importin mtr10

Abstract

Telomerase is a ribonucleoprotein particle (RNP) involved in chromosome end replication, but its biogenesis is poorly understood. The RNA component of yeast telomerase (Tlc1) is synthesized as a polyadenylated precursor and then processed to a mature poly(A)- form. We report here that the karyopherin Mtr10p is required for the normal accumulation of mature Tlc1 and its proper localization to the nucleus. Neither TLC1 transcription nor the stability of poly(A)- Tlc1 is significantly affected in mtr10delta cells. Tlc1 was mostly nuclear in a wild-type background, and this localization was not affected by mutations in other telomerase components. Strikingly, in the absence of Mtr10p, Tlc1 was found dispersed throughout the entire cell. Our results are compatible with two alternative models. First, Mtr10p may import a cytoplasmic complex containing Tlc1 and perhaps other components of telomerase, and shuttling of Tlc1 from the nucleus to the cytoplasm and back may be necessary for the biogenesis of telomerase (the "shuttling" model). Second, Mtr10p may be necessary for the nuclear import of some enzyme needed for the nuclear processing and maturation of Tlc1, and in the absence of this maturation, poly(A)+ Tlc1 is aberrantly exported to the cytoplasm (the "processing enzyme" model).

FIG. 1.

mtr10 mutants have short telomeres. (A) Plasmid pBST3. Two cassettes, each consisting of an HO site followed by a 41-bp-long yeast telomeric sequence (TEL), flanked a copy of the URA3 gene as shown. Induction of the HO endonuclease releases a linear plasmid containing a LEU2 selectable marker. This plasmid can be efficiently maintained only when the exposed telomeric tracts are elongated to create a functional telomere. (B) MTR10 suppressed both the growth defect and the short telomere phenotype of lpm25 mutants. MTR10 was subcloned into pRS314 and transformed into lpm25 cells. Mutants carrying the MTR10 gene were indistinguishable from a wild-type strain. Vector alone did not produce any effect. Loss of MTR10 plasmid from lpm25 strains reverted both phenotypes. Plates were incubated at 30°C for 24 h. Some colonies from these plates were inoculated in liquid medium and grown to an OD₆₀₀ of 1. Genomic DNA from these cultures was used in the Southern blotting. The arrow indicates a band corresponding to Y′ subtelomeric DNA. +, plasmid present; −, plasmid absent.

FIG. 2.

mtr10 cells are defective in the telomerase pathway. (A) Progressive telomere shortening in mtr10Δ mutants. A heterozygous diploid MTR10/mtr10Δ was sporulated and dissected. Spores germinated on YEPD plates for 4 days at 30°C. Wild-type (WT) and mutant strains were selected and then either inoculated in YEPD liquid and grown overnight for genomic DNA isolation (time 0) or streaked out on plates and grown for 24 h. This procedure was repeated for six consecutive days. The Southern blot shows only the Y′ class telomere band and size markers. Telomere length remained constant in a wild-type background throughout the course of the experiment but progressively diminished in the mutant. (B) Overexpression of TLC1 restored wild-type telomere length in the mtr10Δ mutant. Heterozygous diploid MTR10/mtr10Δ cells were transformed with the 2μm plasmid pRS426 or derivatives carrying genes TLC1, EST1, EST2, EST3, and CDC13. After sporulation, dissection, and germination, plasmid-bearing wild-type (WT) and mutant (m) strains were selected and streaked out for four successive days on medium lacking uracil. Again, only the Y′ class telomere band is shown. (C) Low levels of Tlc1 in mtr10Δ cells. Endogenous and overexpression levels of Tlc1, both poly(A)⁺ and mature (mTlc1) forms, were examined by Northern blotting. The level of endogenous mTlc1 was very low in an mtr10Δ mutant, whereas the poly(A)⁺ form was even easier to detect than in the wild-type strain. mTlc1 levels in mtr10Δ cells were greatly restored by overexpression of TLC1 from its own promoter by using a high-copy-number plasmid (pTLC1). By contrast, when overexpressed from the GAL promoter (pSD171), little, if any, mTlc1 accumulated in the mutant. Exposure time was different for the three panels presented. scR1 RNA is shown with identical exposure time. mtr10 mutations did not appreciably influence the levels of this RNA.

FIG. 3.

Normal levels of UsnRNAs in mtr10Δ mutants. Equal amounts of total RNA from five wild-type and five mtr10Δ isogenic strains were analyzed by Northern blotting, using probes specific for U1, U2, U4, U5, scR1, and Tlc1. Quantitation was performed using a PhosphorImager and referred to the levels of scR1. Wild-type levels were assigned a value of 100. The means of the five measures and the standard deviations (error bars) are shown.

FIG. 4.

Telomere length in npl3, hrb1, and gbp2 mutants. Telomere length in single, double, and triple mutants as well as wild-type (WT) strains derived from a heterozygous diploid npl3 hrb1 gbp2/NPL3 HRB1 GBP2 was analyzed by Southern blotting. Cells were streaked out for four successive days before DNA was isolated. For comparison, we also included an mtr10Δ mutant in the same genetic background. Only the Y′-type telomeres and size markers are shown.

FIG. 5.

Defective biogenesis of Tlc1 in mtr10Δ strains. (A) The stability of mTlc1 did not depend on Mtr10 function. tlc1Δ MTR10 and tlc1Δ mtr10Δ strains carrying plasmids pmtr10-7 and pGAL-TLC1 were grown overnight at 18°C in galactose-containing medium. Cultures were shifted to 37°C for 2 h prior to addition of glucose. Samples were taken at the specified times, total RNA was extracted, and equal amounts of RNA were examined by Northern blot analysis. scR1 was probed as a loading reference. Signal intensities for Tlc1 were obtained with different exposures between strains. (B) mTlc1 fails to accumulate in mtr10Δ cells. Strains carrying pGAL-TLC1 were grown overnight (o/n) in raffinose-containing medium, then supplemented with galactose (2% final concentration), and sampled at the specified times. Equal amounts of total RNA from these samples were analyzed by Northern blotting. scR1 was used as a loading control. Samples from overnight-grown cells in galactose-containing medium are also shown (Gal).

FIG. 6.

Tlc1 is mislocalized in mtr10Δ cells. (A) Localization of Tlc1 and U3 in wild-type and mtr10Δ strains. Overexpressed Tlc1 (pRS426-TLC1) and endogenous U3 were visualized with antisense RNA probes labeled with Alexa Fluor 488. tlc1Δ cells served as control for Tlc1-specific detection. U3 localization was nuclear in both backgrounds, whereas Tlc1 dramatically changed its localization in the absence of Mtr10p. Here and below, DAPI-stained DNA is also shown. (B) Localization of galactose-induced Tlc1. Cells were grown in raffinose-containing medium, then split in two, and half were supplemented with 2% galactose for 90 min. Tlc1 was visualized with the same probe as above. (C) Localization of Tlc1 in est mutants. est1Δ, est2Δ, and the quadruple mutant were transformed with pGAL-TLC1 and grown overnight in galactose-containing medium. est3Δ and cdc13^est carried pRS426-TLC1 and were grown in glucose-containing medium. Tlc1 was visualized as described above. Cells carrying pGAL-TLC1 showed no signal when grown in glucose-containing medium (data not shown).

FIG. 7.

Models for the biogenesis of yeast telomerase. (A) Shuttling model. Tlc1 would travel to the cytoplasm where it would probably pick up some proteins. Its nuclear reimport would be mediated by Mtr10p. Once in the nucleus the processing of Tlc1 would continue with the assembly of the Sm proteins, which would trigger the cap hypermethylation and 3′ end trimming. Note that the Sm proteins would follow a different transport pathway (2). (B) Processing enzyme model. Tlc1 may or may not travel to the cytoplasm, but its reimport would not depend on Mtr10p. Instead, Mtr10p is transporting to the nucleus an enzyme performing an essential function for proper telomerase biogenesis. A₈₀ denotes the poly(A)⁺ tail of Tlc1 (≈80 nt long) (4).