Biology subjects

Shippen, D. E.

Publications and source records attributed to Shippen, D. E..

3 recordsLinked to original sources

bioRxiv2026

La1: an evolutionarily conserved player in the Arabidopsis telomerase complex

Analysis of yeast and mammalian telomerase ribonucleoprotein (RNP) complexes reveals striking divergence in their biogenesis and protein complements. However, little is known about telomerase in plants. In addition to the catalytic subunit TERT and the templating RNA TR, we previously reported Arabidopsis thaliana contains two telomerase accessory factors, AtNAP57, a dyskerin homolog that in mammals is essential for telomerase activity, and the telomeric DNA binding protein AtPOT1a. Both proteins stimulate Arabidopsis telomerase repeat addition processivity. Here we employ quantitative mass spectrometry (MS) to further examine telomerase in Arabidopsis. Unexpectedly, dyskerin and AtPOT1a were not detected in our purified complexes, but AtLa1, an RNA-binding factor that recognizes the UUU-3'OH of RNA Pol III transcripts, was highly enriched. RNA-IP assays confirmed AtLa1 association with AtTR in vivo. RNAi-mediated knockdown of AtLa1 strongly diminished telomerase activity, indicating AtLa1 is required for its function in vivo. In vitro binding studies revealed that AtLa1 contacts AtTR via the UUU-3'OH and a plant-specific P1a-P1b-P4 three-way junction (TWJ). Since the TWJ is also required for AtNAP57 binding, the data suggesting that AtNAP57 and AtLa1 compete for AtTR binding or sequentially associate during RNP during biogenesis. In contrast to AtNAP57, AtLa1 did not stimulate telomerase activity when TERT and TR were assembled in vitro, consistent with function during a different step in telomerase assembly. We conclude Arabidopsis telomerase employs multiple accessory factors utilized by both mammalian and single-celled relatives. Further exploration of Arabidopsis telomerase may offer novel insight into telomerase evolution and mechanisms of biogenesis. Significance of ResearchQuantitative mass spectrometry of Arabidopsis telomerase uncovered AtLa1, a homolog of ciliate and yeast proteins that promotes telomerase maturation. AtLa1 is essential for telomerase function in vivo, and in vitro it engages the same region of AtTR bound by AtNAP57, homologous to a telomerase accessory from mammals.

plant biology↗

bioRxiv2019

Chromatin organization in early land plants reveals an ancestral association between H3K27me3, transposons, and constitutive heterochromatin

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes and our knowledge of the evolution of chromatin related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants 400 Mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional organization of the genome of early land plants. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and repeats in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres, and a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that chromatin regions belonging to repressed heterochromatin were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

molecular biology↗

bioRxiv2019

A hypomorphic allele of telomerase reverse transcriptase uncovers the minimal functional length of telomeres in Arabidopsis.

Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species differs by up to four orders of magnitude, raising the question of the minimal length of telomeric DNA necessary for proper function. Here we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.

genetics↗