Telomeres and their associated factors in Arabidopsis thaliana

Telomeres are important protein-DNA structures at the ends of linear eukaryotic chromosomes that are necessary for genome integrity. Telomeres are maintained by intermittent action of telomerase. I explored the kinetics of telomere length homeostasis in the model plant Arabidopsis thaliana by crossing wild type plants to different generations of telomerase deficient plants, and then analyzing telomere length in the resulting progeny. Unexpectedly, I found plants lacking telomerase for seven generations can lengthen telomeres when telomerase is reintroduced, but one generation is not sufficient to reestablish the telomere set point. Est1 is a non-catalytic component of the Saccharomyces cerevisiae telomerase holoenzyme. To investigate the role of Est1 in higher eukaryotes, I identified two putative Est1 homologues in Arabidopsis, AtEST1a and AtEST1b. Plants deficient in AtEST1a displayed no vegetative or reproductive defects. However, plants deficient for AtEST1b were sterile and had severe vegetative and reproductive irregularities. Surprisingly, no defects in telomere maintenance were observed in any single or double mutant line. This suggests that the Est1- like proteins in plants have evolved new functions outside of telomere length maintenance and end protection.One consequence of telomere dysfunction is end-to-end chromosome fusion. In mammals, telomere fusion is mediated through NHEJ and requires DNA Ligase IV (Lig4). Lig4 is an essential component of the NHEJ pathway along with the Ku70/Ku80 heterodimer and DNA-PKcs. To address the mechanism of chromosome fusion in Arabidopsis, we investigated the role of Lig4 in mutant combinations lacking TERT, the catalytic subunit of telomerase, and Ku70. Surprisingly, telomere end-to-end fusions were observed in ku70 tert lig4 triple mutants, suggesting that neither Lig4 nor Ku70 are required for the fusion of critically shortened telomeres in Arabidopsis. To investigate the origin of genome instability, terminal restriction fragment analysis was performed on triple mutants. Strikingly, telomeres diminished five to six-fold faster than in a tert single mutant. Moreover, in the triple mutants, telomere tracts were extremely heterogeneous, suggesting that the telomeres were exposed to catastophic nucleolytic attack. These data provide the first evidence that Lig4 contributes to telomere maintenance and chromosome end protection

Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV

In the absence of the telomerase, telomeres undergo progressive shortening and are ultimately recruited into end-to-end chromosome fusions via the non-homologous end joining (NHEJ) double-strand break repair pathway. Previously, we showed that fusion of critically shortened telomeres in Arabidopsis proceeds with approximately the same efficiency in the presence or absence of KU70, a key component of NHEJ. Here we report that DNA ligase IV (LIG4) is also not essential for telomere joining. We observed only a modest decrease (3-fold) in the frequency of chromosome fusions in triple tert ku70 lig4 mutants versus tert ku70 or tert. Sequence analysis revealed that, relative to tert ku70, chromosome fusion junctions in tert ku70 lig4 mutants contained less microhomology and less telomeric DNA. These findings argue that the KU-LIG4 independent end-joining pathway is less efficient and mechanistically distinct from KU-independent NHEJ. Strikingly, in all the genetic backgrounds we tested, chromosome fusions are initiated when the shortest telomere in the population reaches ∼1 kb, implying that this size represents a critical threshold that heralds a detrimental structural transition. These data reveal the transitory nature of telomere stability, and the robust and flexible nature of DNA repair mechanisms elicited by telomere dysfunction

Telomere dynamics and fusion of critically shortened

telomeres in plants lacking DNA ligase I

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA

Neurologic abnormalities in mouse models of the lysosomal storage disorders mucolipidosis II and mucolipidosis III γ.

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II) or the more moderate mucolipidosis III alpha/beta (ML III α/β), while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ). Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments

<i>Gnptab^−/−</i> mice exhibit performance deficits on sensorimotor and rotarod tests which show progressive impairment with age.

<p>(A–D) At 1-month of age, the <i>Gnptab^−/−</i> mice spent significantly (*<i>p</i> = 0.026) less time on an elevated platform (A), took significantly (*<i>p</i>  =  0.0007) longer to climb down a pole (B), and spent significantly (*<i>p</i>  =  0.020) less time hanging upside down on an inverted screen (C) compared to WT littermate control mice. The <i>Gnptab^−/−</i> mice also showed a nonsignificant trend in terms of spending less time on an elevated ledge (D). (E-H) Larger performance deficits were observed when the mice were tested at 4-5 months of age when significant impairment was observed in the <i>Gnptab^−/−</i> group compared to WT controls on the platform (E; *<i>p</i>  =  0.0002), pole (F; *<i>p</i>  =  0.0005), inverted screen (G; *<i>p</i> <0.00005), and ledge (H; *<i>p</i>  =  0.001) tests (see Fig. 3A for details of sex effects on the ledge test). (I-K) The <i>Gnptab^−/−</i> mice demonstrated mild performance impairments on the rotarod when they were tested at 1-month of age. (I) For example, the <i>Gnptab^−/−</i> mice spent significantly less time on the stationary rod component of the test compared to the WT group (genotype effect, ††<i>p</i>  =  0.002); genotype x trials interaction (**<i>p</i>  =  0.0004), but this was mostly due to differences observed on trial 1 (*<i>p</i>  =  0.0007). (J) Analysis of the constant speed rotarod data showed that large differences were observed between the <i>Gnptab^−/−</i> and WT groups but only during the first session (genotype x sessions interaction; **<i>p</i>  =  0.024); trial 1 (†<i>p</i>  =  0.046); trial 2 (*<i>p</i>  =  0.022). (K) No significant effects involving Genotype were found as a result of analyzing the accelerating rotarod data. (L-M) The <i>Gnptab^−/−</i> mice showed severely impaired performance on the rotarod tasks when tested at 4–5 months of age. Specifically, the <i>Gnptab^−/−</i> mice were significantly impaired on the stationary rod (L; genotype effect: **<i>p</i> <0.00005; pairwise comparisons for each trial: *<i>p</i> <0.0005), the constant speed rotarod (M; genotype effect: **<i>p</i> <0.00005; pairwise comparisons for each trial: *<i>p</i> <0.003), and accelerating rotarod (N; genotype effect: ***<i>p</i> <0.00005; genotype x trials interaction: **<i>p</i> <0.010; pairwise comparisons for each trial: *<i>p</i> <0.003).</p

<i>Gnptg^−/−</i> mice show mild to moderate performance deficits on activity, sensorimotor and rotarod tests.

<p>(A-B) Although the <i>Gnptg^−/−</i> mice tended to show lower levels of ambulatory activity (A) and vertical rearing frequency (B) relative to WT littermate controls on the 1-h locomotor activity test when tested at 4–6 months of age, no significant effects involving genotype were found following analyses conducted on these data. However the <i>Gnptg^−/−</i> group took significantly longer to climb to the top of the 60° (C) and 90° (D) inclined screens, (genotype effects: *<i>p</i>  =  0.0005 and 0.0001, respectively), and showed a nonsignificant trend toward being able to remain on the platform for a shorter time compared to WT control mice (E) when tested at this age (see Fig. 3B for details of sex effects on the 60° inclined screen). (F) When the 1-h activity test was conducted at 12–14 months of age, the <i>Gnptg^−/−</i> mice tended to be less active compared to controls, although this depended on the time block of the test session (genotype x time interaction: *<i>p</i>  =  0.012), with the largest group differences being observed during the first time block (†<i>p</i>  =  0.041). (G) At this age, the <i>Gnptg^−/−</i> group also showed significantly reduced rearing frequency on average across time blocks, (genotype effect: *<i>p</i>  =  0.043), with the largest differences occurring during the first block (†<i>p</i>  =  0.049). (H) During testing at 12-14 months of age, the performance level of the <i>Gnptg^−/−</i> mice tended to be much lower than that of the WT control group on the 60° inclined screen, but the differences were not statistically significant. However, the <i>Gnptg^−/−</i> mice did show significant performance deficits at this age on the 90° inclined screen (I) and platform tests (J), (genotype effects: *<i>p</i>  =  0.026 and *<i>p</i>  =  0.010, respectively). The <i>Gnptg^−/−</i> strain was also impaired on the rotarod when tested at 4–6 months of age. (K) For example, a significant genotype effect (*<i>p</i>  =  0.024) indicated that the <i>Gnptg^−/−</i> mice were impaired on the stationary rod component of the rotarod although this effect was mostly due to differences observed during trial 1 (††<i>p</i>  =  0.007), whereas their performance on the other two trials were similar to those of the WT group (see Fig. 3C for details of sex effects). (L) The <i>Gnptg^−/−</i> group also exhibited significant performance impairments on the constant speed rotarod on average across trials and sessions (genotype effect: *<i>p</i>  =  0.005), with group differences being significant for session 1 - trial 2 (**<i>p</i>  =  0.004), while large differences were also observed for session 2 - trial 2 (†<i>p</i>  =  0.044) and session 3 - trial1 (††<i>p</i>  =  0.019). (M) Analysis of the accelerating rotarod data also revealed significant performance deficits on the part of the <i>Gnptg^−/−</i> mice (genotype effect: **<i>p</i>  =  0.0003; genotype x sessions interaction: *<i>p</i>  =  0.002), although this was somewhat dependent on the sessions variable. Pair-wise comparisons showed that group performances differed significantly for session 1 - trial 2, session 2 - trial 2, and session 3 - trial 1 (***<i>p</i> <0.0009), while large differences were observed for session 2 - trial 1 (†<i>p</i>  =  0.012) and session 3 - trial 2 (††<i>p</i>  =  0.009). (See Fig. 3D for details concerning sex effects.) When the mice were re-tested at 12-14 months, the groups were found to perform similarly on the stationary rod (N). However, the <i>Gnptg^−/−</i> group again showed significant deficits on the constant speed rotarod (O) which was documented by a significant genotype effect (**<i>p</i>  =  0.0003). Pair-wise comparisons showed that the <i>Gnptg^−/−</i> mice had significantly reduced times on the rod for session 1 - trial 2 (***<i>p</i>  =  0.0003) and session 2 - trial 2 (*<i>p</i>  =  0.004), with large differences also being found for session 2 - trial1 (†<i>p</i>  =  0.033). (P) The <i>Gnptg^−/−</i> group was also significantly impaired again on the accelerating rotarod when re-tested at the later age, (genotype effect: **<i>p</i>  =  0.0001) when pair-wise comparisons showed significant group differences across all trials and sessions (*<i>p</i> <0.008; ***<i>p</i> <0.0005) (see Fig. 3E for details of sex effects).</p

PAS and IHC staining of brain and spinal cord dystrophic axons and neurites of 12-month-old <i>Gnptab^−/−</i> mice.

<p>(A) The dystrophic axons and neurites were consistently negative for PAS (arrows). (B) There was widespread granular staining of most dystrophic axons/neurites for ubiquitin (arrows). In contrast, IHC staining for the autophagy marker LC3B (C) and the lysosome/endosome marker LAMP-1 (D) showed variable (negative to mild) staining of dystrophic axons/neurites (arrows). (E) Strong staining for <i>neurofilament protein</i> (NFP) was present in only a few dystrophic axons in the white tracts of the cerebellum and spinal cord of <i>Gnptab^−/−</i> mice, while the abundant large swollen neurites in the superficial laminae (layers 1 to 3) of the dorsal horn grey matter were consistently NFP-negative (arrows). (40X; Bar  =  100 mm)</p