RESUMO
KEY MESSAGE: tRNA Adenosine Deaminase 3 helps to sustain telomere tracts in a telomerase-independent fashion, likely through regulating cellular metabolism. Telomere length maintenance is influenced by a complex web of chromatin and metabolism-related factors. We previously reported that a lncRNA termed AtTER2 regulates telomerase activity in Arabidopsis thaliana in response to DNA damage. AtTER2 was initially shown to partially overlap with the 5' UTR of the tRNA ADENOSINE DEAMINASE 3 (TAD3) gene. However, updated genome annotation showed that AtTER2 was completely embedded in TAD3, raising the possibility that phenotypes ascribed to AtTER2 could be derived from TAD3. Here we show through strand-specific RNA-Seq, strand-specific qRT-PCR and bioinformatic analyses that AtTER2 does not encode a stable lncRNA. Further examination of the original tad3 (ter2-1/tad3-1) mutant revealed expression of an antisense transcript driven by a cryptic promoter in the T-DNA. Hence, a new hypomorphic allele of TAD3 (tad3-2) was examined. tad3-2 mutants showed hypersensitivity to DNA damage, but no deregulation of telomerase, suggesting that the telomerase phenotype of tad3-1 mutants reflects an off-target effect. Unexpectedly, however, tad3-2 plants displayed progressive loss of telomeric DNA over successive generations that was not accompanied by alteration of terminal architecture or end protection. The phenotype was exacerbated in plants lacking the telomerase processivity factor POT1a, indicating that TAD3 promotes telomere maintenance through a non-canonical, telomerase-independent pathway. The transcriptome of tad3-2 mutants revealed significant dysregulation of genes involved in auxin signaling and glucosinolate biosynthesis, pathways that intersect the stress response, cell cycle regulation and DNA metabolism. These findings indicate that the TAD3 locus indirectly contributes to telomere length homeostasis by altering the metabolic profile in Arabidopsis.
Assuntos
Adenosina Desaminase/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA de Plantas/genética , Telômero/genética , Regiões 3' não Traduzidas , Adenosina Desaminase/metabolismo , Apoptose/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Dano ao DNA , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , Análise de Sequência de RNA , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero/genética , Homeostase do Telômero/fisiologiaRESUMO
Telomerase is essential for maintaining telomere integrity. Although telomerase function is widely conserved, the integral telomerase RNA (TR) that provides a template for telomeric DNA synthesis has diverged dramatically. Nevertheless, TR molecules retain 2 highly conserved structural domains critical for catalysis: a template-proximal pseudoknot (PK) structure and a downstream stem-loop structure. Here we introduce the authentic TR from the plant Arabidopsis thaliana, called AtTR, identified through next-generation sequencing of RNAs copurifying with Arabidopsis TERT. This RNA is distinct from the RNA previously described as the templating telomerase RNA, AtTER1. AtTR is a 268-nt Pol III transcript necessary for telomere maintenance in vivo and sufficient with TERT to reconstitute telomerase activity in vitro. Bioinformatics analysis identified 85 AtTR orthologs from 3 major clades of plants: angiosperms, gymnosperms, and lycophytes. Through phylogenetic comparisons, a secondary structure model conserved among plant TRs was inferred and verified using in vitro and in vivo chemical probing. The conserved plant TR structure contains a template-PK core domain enclosed by a P1 stem and a 3' long-stem P4/5/6, both of which resemble a corresponding structural element in ciliate and vertebrate TRs. However, the plant TR contains additional stems and linkers within the template-PK core, allowing for expansion of PK structure from the simple PK in the smaller ciliate TR during evolution. Thus, the plant TR provides an evolutionary bridge that unites the disparate structures of previously characterized TRs from ciliates and vertebrates.