Abstract
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 RESEARCH: Quantitative 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.