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Identification of Purple Sea Urchin Telomerase RNA Using a Next-Generation Sequencing Based Approach

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Identification of Purple Sea Urchin Telomerase RNA Using a Next-Generation Sequencing Based Approach Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach YANG LI,1,6 JOSHUA D. PODLEVSKY,2,6 MANJA MARZ,3,6 XIAODONG QI,1 STEVE HOFFMANN,4,5 PETER F. STADLER,5 and JULIAN J.-L. CHEN1,7 1Department of Chemistry & Biochemistry and 2School of Life Sciences, Arizona State University, Tempe, Arizona 85287, USA 3Department of Bioinformatics, Friedrich Schiller University of Jena, D-07743 Jena, Germany 4LIFE Center and 5Interdisciplinary Center for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany ABSTRACT Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms. Here we report a novel approach for TR discovery that combines in vitro TR enrichment from total RNA, next-generation sequencing, and a computational screening pipeline. With this approach, we have successfully identified TR from Strongylocentrotus purpuratus (purple sea urchin) from the phylum Echinodermata. Reconstitution of activity in vitro confirmed that this RNA is an integral component of sea urchin telomerase. Comparative phylogenetic analysis against vertebrate TR sequences revealed that the purple sea urchin TR contains vertebrate-like template-pseudoknot and H/ACA domains. While lacking a vertebrate- like CR4/5 domain, sea urchin TR has a unique central domain critical for telomerase activity. This is the first TR identified from the previously unexplored invertebrate clade and provides the first glimpse of TR evolution in the deuterostome lineage. Moreover, our TR discovery approach is a significant step toward the comprehensive understanding of telomerase RNP evolution. Keywords: telomere; telomerase RNA; ribonucleoprotein; next-generation sequencing INTRODUCTION gal and vertebrate TRs in the CR4/5 (vertebrate) and three- way-junction (fungal) domains (Qi et al. 2013). However, Telomerase is a specialized reverse transcriptase (RT) essential the critical loop of P6.1 element is not conserved in closely for thesynthesisof telomeric DNA repeatsonto the ends oflin- related budding yeasts. Furthermore, these universally con- ear chromosomes. Functioning as a ribonucleoprotein (RNP) served structural elements have yet to be found within the re- enzyme, telomeraseconsistsoftwoessentialcorecomponents: cently identified Arabidopsis TR (Cifuentes-Rojas et al. 2011). the catalytic telomerase reverse transcriptase (TERT) protein The telomerase holoenzyme comprises a myriad of accessory and telomerase RNA (TR) that provides the template for proteins that vary dramatically among species by binding DNA synthesis (Blackburn and Collins 2011). TERT is highly species-specific structural domains within TR. These acces- conserved, with the central domain constituting the active site sory proteins are essential for regulating telomerase biogen- and an N-terminal domain that binds specifically to the TR esis. Understanding the evolution of telomerase structure (Bleyet al. 2011; Mason et al. 2011). In contrast, TR isextreme- and function requires the identification of TR sequences ly divergent in size, sequence, and structure (Podlevsky et al. from all major clades. However, the massive divergence in 2008; Podlevsky and Chen 2012). TR sequence hinders TR discovery from unexplored phyloge- Ciliate, yeast, and vertebrate TRs show no sequence ho- netic groups by conventional molecular and bioinformatics mology or structural similarity apart from the universal pseu- approaches. doknot structure. Following the identification of Neurospora Molecular biology approaches for TR identification have crassa and 72 additional filamentous fungal TRs, a second relied heavily on telomerase enzyme purification (Greider highly conserved element, P6/6.1, was identified within fun- and Blackburn 1989; Leonardi et al. 2008; Webb and Zakian 2008; Cifuentes-Rojas et al. 2011; Qi et al. 2013). However, 6These authors contributed equally to this work. telomerase purification is not feasible for the vast majority 7Corresponding author of organisms due to the low abundance of the enzyme or E-mail [email protected] Article published online ahead of print. Article and publication date are at the lack of genetic tools necessary for expressing recombinant http://www.rnajournal.org/cgi/doi/10.1261/rna.039131.113. affinity-tagged proteins. Even within species amenable to 852 RNA (2013), 19:852–860. Published by Cold Spring Harbor Laboratory Press. Copyright © 2013 RNA Society. Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press A new approach for telomerase RNA identification telomerase purification, this methodology is often complex, RESULTS requiring multiple purification steps and compatible telome- Cloning TR from invertebrate species has been hindered by rase activity assays to follow telomerase activity throughout the low abundance of telomerase enzyme in cells, as well as purification. the size, sequence, and structural divergence of TR. To over- Bypassing enzyme purification from cells, PCR amplifica- come these obstacles and create a simple and straightfor- tion from genomic DNA has been successfully applied for ward system for identifying TRs, we have developed a novel TR identification from a large number of vertebrate species approach that uses in vitro enrichment of the TR from (Chen et al. 2000). Based on the human TR sequence, degen- total RNA, next-generation sequencing, and bioinformatics erate PCR primers targeted the conserved pseudoknot and screening. In this study, we aimed to identify the TR from S. CR4/5 regions. This method is not effective for certain spe- purpuratus, a model organism that has a sequenced genome cies, such as teleost fishes and some reptiles, where the primer and is closely related to vertebrates. The multiple-step TR-en- targeting sites are not well conserved. Alternatively, PCR richment strategy we developed is based on the assumptions amplification using primers targeting syntenic protein genes ′ that invertebrate TR would have a 5 -trimethylguanosine flanking the TR gene has been used to identify TRs from (TMG) cap as well as preferentially be bound by the TERT Sensu Stricto Saccharomyces species (Dandjinou et al. 2004). protein in vitro. These are two common features of vertebrate However, these PCR-based methodologies are most adept and yeast TRs (Seto et al. 1999; Jady et al. 2004; Webb and for a group of species in which an initial TR sequence has Zakian 2008). A cDNA library generated from the enriched been identified previously. In place of PCR amplifying from RNA was then subjected to next-generation sequencing, fol- genomic DNA, RT-PCR amplifying the TR transcript from lowed by in silico assembly of sequencing reads, screening total RNA with primers targeting the template sequence pre- for putative template sequences, and removal of known or hy- dicted from telomeric DNA repeat sequence has been used pothetical gene candidates. to identify TR transcripts from a number of Candida yeast TR enrichment was performed in three sequential steps: species that contain relatively long TR templates (Gunisova anti-TMG immuno-precipitation (IP), Terminator exonucle- et al. 2009). Other molecular methods including nucleic ase treatment and TERT co-IP of RNA (Fig. 1). In the first step acid hybridization and/or gene-knockout library screening ofTRenrichment,12mgofinputtotalRNAisolatedfrompur- have thus far been applied exclusively in yeasts for TR identi- ple sea urchin gonads produced 5 µg of TMG-capped RNA. fication (McEachern and Blackburn 1995; Hsu et al. 2007; Next, this TMG-capped RNA was treated with Terminator, a Kachouri-Lafond et al. 2009). These other molecular ap- ′ 5 -monophosphate-dependent exonuclease (Epicentre). The proaches are generally limited to organisms with small ge- TMG-capped RNAs were resistant to Terminator exonuclease nomes and established genetic tools, and cannot be readily degradation while copurified ribosomal RNAs (rRNAs), applied to other groups of species. ′ which contain a 5 -monophosphate, were degraded. The Recent advances in genome sequencing have made BLAST, final step of TR enrichment was TERT co-IP of RNA. A basic local alignment search tool, a rather straightforward means for searching TR homologs within closely related spe- cies (Altschul et al. 1990; Qi et al. 2013). Augmenting BLAST with position-specific weight matrices (PWM) has been shown to enhance degenerate TR sequence searches in teleost fish (Xie et al. 2008). While PWM allows for more degenerate search patterns, a sufficient number of well-aligned sequences are required to generate the weight matrices for nucleotide probability at each position (Mozig et al. 2007). Thus, this methodology is most applicable for TR identification from species diverged from a closely related group for which nu- merous sequences have previously been identified. To date, no TR has been identified from invertebrates despite their evolutionary proximity to vertebrates. Here we report the identification of the first invertebrate TR from Strongylocentrotus purpuratus (purple sea urchin) using a novel approach that combines in vitro TR enrichment from total RNA, next-generation
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