trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase

Abstract

Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9-1-1) proliferating cell nuclear antigen-like sliding clamp. Thus, the 9-1-1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1.

Figure 1. trt-1 Is the C. elegans Catalytic Subunit of Telomerase

(A) Three X-autosome fusions isolated from trt-1(e2727) display linkage between an end of the X chromosome and an end of an autosome. ypT3 and ypT16 were independent chromosome fusions isolated from parallel lines grown from the same F2 founder, suggesting that the two fusiogenic chromosome ends may correspond to short telomeres in the F2 founder, recapitulating our previous observations regarding strains inheriting a short left end of the X chromosome in mrt-2 mutants [12] and supporting a mouse study of telomere length and genome instability in the absence of the telomerase RNA [61]. (B) End-to-end fusions in oocytes of late-generation trt-1 mutant worms. Two DAPI-stained oocyte nuclei are shown per picture as indicated by dashed circles. (C) trt-1 gene structure with mutations. Exon-intron structures and TERT protein domains are shown. Effects of mutations for all four trt-1 alleles are shown as determined by genomic DNA and cDNA sequencing. Black boxes are used to depict exons missing as a consequence of trt-1 splice-junction mutations or sequences eliminated by trt-1 deletions. For yp1, intron 6 is retained and a cryptic intron in exon 7 becomes active. Sequences flanking each breakpoint are shown for trt-1 deletions. A 13-bp in-frame insertion occurred at the breakpoint of trt-1(ok410). Sequencing of cDNA from trt-1(tm899) indicated that the deletion results in a premature stop codon.

Figure 2. Progressive Telomere Shortening in trt-1 Mutants and Rescue by the Wild-Type DY3.4 Gene

(A) The trt-1 alleles tm899 and ok410 displayed comparable rates of telomere shortening over progressive generations. Two generations of N2 wild-type are shown for comparison. Southern blotting was performed using a (TTAGGC)_n probe and genomic DNA prepared from propagated strains as described [12]. (B and C) Expression of DY3.4 on the ypEx1 extrachromosomal array (B) eliminated telomere shortening for trt-1(ok410) in comparison with a nonrescued control and (C) repressed the formation of chromosome fusions observed in late-generation ok410 and e2727 mutants. Adult animals were stained with DAPI at generation F10, where nonrescued siblings exhibited a strongly reduced brood size or sterility. For Southern blots, sizes of DNA fragments are indicated to the left (kb).

Figure 3. Structure and Sequence Motifs of Caenorhabditis TERT Proteins

(A) Structure of TERT proteins from distantly related species, with isoelectric point (pI) indicated. (B) TERT sequence motifs include seven canonical reverse transcriptase motifs (1, 2, A, B′, C, D, and E) and several TERT-specific N-terminal motifs (I/DAT/GQ, II/CP, and T) [–20] for TERT proteins from three Caenorhabditis species; C. elegans (Ce), C. briggsae (Cb), and C. remanei (Cr); from the vertebrates H. sapiens (Hs) and Fugu rubripes (Fr); and from Saccharomyces cerevisiae (Sc) and Tetrahymena thermophila (Tt). An additional motif T is included for the nematode Brugia malayi (Bm), as this sequence was critical for precisely defining the Caenorhabditis motif T. Only portions of motif I/GQ and motif DAT with homology to the Caenorhabditis TERT proteins are shown. Motif DAT is a specialized protein domain within motif I/GQ that is known to be required for telomerase activity in vivo [42]. A clearly defined III/QFP N-terminal motif was not identified for the Caenorhabditis TERTs. The Brugia malayi T motif is based on a short expressed sequence tag, and further N-terminal motifs could not be discerned from the current genome sequence (http://www.tigr.org). Sequence alignments were performed using Pole BioInformatique Lyonnais ClustalW 1.8 (http://pbil.ibcp.fr/htm/index.php).

Figure 4. trt-1 and mrt-2 Function in the Same Pathway

(A) Progressive telomere shortening of mrt-2 single, trt-1(tm899) single, and trt-1(tm899);mrt-2 double mutant strains. (B) Comparison of late onset of sterility in mrt-2 single, trt-1(tm899) single and trt-1(tm899);mrt-2 double mutant strains. Two independent strains, a and b, are shown for each genotype. Plates were seeded with six L1 animals, and the brood size was scored after 7 days of growth as wild-type (W), medium (M), few (F), and sterile (S) [12]. Note that initial telomere lengths observed for these strains reflect random telomere segregation during a cross. Therefore, the rate of telomere erosion is a more significant measure of additivity than are differences in initial telomere length or the time to sterility. Sizes of DNA fragments are indicated to the right (kb).

Figure 5. trt-1 Mutants Display Mitotic Defects at Sterility

(A) Wild-type germlines displayed two symmetric, highly proliferative arms of the germline when stained with DAPI. (B–K) In contrast, trt-1 mutants at sterility displayed medium and small germline arms (B), small and empty germline arms (C), and only empty germline arms (D). Some trt-1 mutants had a Masculinization of the Germline (Mog) phenotype and only produced sperm (E and F), many displayed endomitotic oocytes that endoreduplicate in the absence of sperm (G), some germlines displayed an abnormal Germline Proliferation (Glp) phenotype where the few germ cells that were produced were mitotic (H and I), and anaphase bridges were observed between intestinal nuclei in sterile trt-1 adults (J and K) but not in wild-type controls. (L) Quantification of somatic and germline phenotypes in wild-type, in trt-1 mutants, and in the kinetochore protein mutant him-10 grown at the restrictive temperature of 25 °C [40]. Phenotypes scored were Protruding Vulva (Pvl), Vulvaless (Vul), Egg-Laying Defective (Egl), Intestinal Nuclear Bridge (Ibr), Abnormal Oocyte Formation (Ooc), Endomitotic Oocyte (Emo), Mog, and Glp. Although the Pvl phenotype was not observed in him-10 animals, it is commonly observed for depletion of other kinetochore components by RNA interference (http://www.wormbase.org).

Figure 6. trt-1 Mutants Have a Normal Life Span Independent of Telomere Length and Telomere Uncapping

(A) Life spans of N2 wild-type and early, middle, and late generations of a trt-1(ok410) strain. Mutants were analyzed at generations with slightly shortened telomeres (F7), short telomeres (F15), and very short telomeres with chromosome fusions (F19). (B) Life spans of N2 wild-type and early and late generations of a trt-1(tm899) strain.