Podlevsky JD et al. (2016), Structure and function of echinoderm telomerase...

ECB-ART-44358

RNA 2016 Feb 01;222:204-15. doi: 10.1261/rna.053280.115.

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Structure and function of echinoderm telomerase RNA.

Podlevsky JD , Li Y , Chen JJ .

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Telomerase is a ribonucleoprotein (RNP) enzyme that requires an integral telomerase RNA (TR) subunit, in addition to the catalytic telomerase reverse transcriptase (TERT), for enzymatic function. The secondary structures of TRs from the three major groups of species, ciliates, fungi, and vertebrates, have been studied extensively and demonstrate dramatic diversity. Herein, we report the first comprehensive secondary structure of TR from echinoderms-marine invertebrates closely related to vertebrates-determined by phylogenetic comparative analysis of 16 TR sequences from three separate echinoderm classes. Similar to vertebrate TR, echinoderm TR contains the highly conserved template/pseudoknot and H/ACA domains. However, echinoderm TR lacks the ancestral CR4/5 structural domain found throughout vertebrate and fungal TRs. Instead, echinoderm TR contains a distinct simple helical region, termed eCR4/5, that is functionally equivalent to the CR4/5 domain. The urchin and brittle star eCR4/5 domains bind specifically to their respective TERT proteins and stimulate telomerase activity. Distinct from vertebrate telomerase, the echinoderm TR template/pseudoknot domain with the TERT protein is sufficient to reconstitute significant telomerase activity. This gain-of-function of the echinoderm template/pseudoknot domain for conferring telomerase activity presumably facilitated the rapid structural evolution of the eCR4/5 domain throughout the echinoderm lineage. Additionally, echinoderm TR utilizes the template-adjacent P1.1 helix as a physical template boundary element to prevent nontelomeric DNA synthesis, a mechanism used by ciliate and fungal TRs. Thus, the chimeric and eccentric structural features of echinoderm TR provide unparalleled insights into the rapid evolution of telomerase RNP structure and function.

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Genes referenced: LOC115922213 tert

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	FIGURE 1. Identification of echinoderm TRs. Eight sea urchins, a sand dollar (Dendraster excentricus), three brittle star, and a feather star (Anneissia japonica) TR species were identified in this study (black text) with two sea urchin TRs (violet text) previously identified (Li et al. 2013; Gillard et al. 2014). The TR length was determined by 5′- and 3′-RACE, estimated from the position of the box ACA in the sequencing data (*), or not determined (n/d). Echinoderms comprise the sister classes Echinoidea (sea urchins and sand dollars), Ophiuroidea (brittle stars), and the ancestral Crinoidea class (sea lilies and feather stars). The vertebrate lineage (phylum Chordata, blue) is closely related to the echinoderm lineage (light violet), while fungi (phylum Ascomycota, green) are the out-group.
	FIGURE 2. Echinoderm TR template/pseudoknot domain contains a template-adjacent helix for template boundary definition. Comparison of representative TR template/pseudoknot domains from vertebrate, human (A); sea urchin, purple sea urchin (B); brittle star, blunt spined brittle star (C); and feather star, Japanese feather star (D). The hallmark triple helix within the TR pseudoknot (green) is denoted within each structure. Covariation (black bar), universal-invariant between vertebrates and echinoderm (red), and group-invariant (orange) nucleotides for vertebrate, sea urchin, and brittle star TR template/pseudoknot domains are based on multiple sequence alignment of 42 vertebrate (Chen et al. 2000; Podlevsky et al. 2008; Xie et al. 2008), nine sea urchin/sand dollar, three brittle star, and a feather star species (see Supplemental Fig. S2). (E–G, top) Schematic of the purple sea urchin TR template/pseudoknot domain denoting nucleotide mutations (violet), insertions (green), and truncations to determine P1.1 functionality for template definition. (E–G, bottom) Functional analysis of purple sea urchin TR mutations and truncation variants by the direct primer-extension assay. Sea urchin TR template/pseudoknot variants (fragment 11–163 nt) and the central domain fragment (186–456 nt) were assembled in vitro with the sea urchin TERT protein. Sea urchin telomerase variants were assayed with the 18-mer DNA primer (TTAGGG)3 and this primer 32P end-labeled was added as a loading control (l.c.). The incorporation of nontelomeric nucleotides from the downstream-flanking region of the template, read-through, is denoted on the gel (red triangles).
	FIGURE 3. Structural divergence in the essential central domain between vertebrate, sea urchin, brittle star, and feather star TRs. Comparison of representative TR central domains from vertebrate, human (A); sea urchin, purple sea urchin (B); brittle star, blunt spined brittle star (C); and feather star, Japanese feather star (D). The minimal functional element for the stimulation of telomerase activity in vertebrates is CR4/5 (open box). Covariation (black bar) and group-invariant (orange) nucleotides for vertebrate, sea urchin, and brittle star TR central domains based on multiple sequence alignment of 42 vertebrate (Chen et al. 2000; Podlevsky et al. 2008; Xie et al. 2008), 10 sea urchin/sand dollar, 3 brittle star, and a feather star species (see Supplemental Fig. S3A,C). The purple sea urchin and blunt spined brittle star central domains were analyzed by SHAPE with flexibility (high, dark blue; low, light blue circles) and rigidity (no circle) of each residue denoted on the secondary structure (see Supplemental Fig. S3B,D).
	FIGURE 4. The echinoderm eCR4/5 is a functional homolog of the vertebrate CR4/5 domain. (A,C) Schematic of the purple sea urchin TR central domain denoting nucleotide deletions (black shaded) and truncations to determine the minimal functional fragment sufficient for the stimulation of telomerase activity. Apical loop truncations were capped with a GNRA tetraloop (gray). The echinoderm eCR4/5 (dashed box), a functional homolog of vertebrate CR4/5, is denoted. (B,D,E) Functional analysis of purple sea urchin TR mutations and truncation variants by the direct primer-extension assay. The purple sea urchin TR template/pseudoknot (PK) fragment (11–163 nt) and central domain variants were assembled in vitro with the sea urchin TERT protein. Sea urchin telomerase variants were assayed with the 18-mer DNA primer (TTAGGG)3 and this primer 32P end-labeled was added as a loading control (l.c.). Relative activity of purple sea urchin TR variants compared against telomerase reconstituted with purple sea urchin TR PK and central domain RNA fragments is denoted below the gel.
	FIGURE 5. The vertebrate TR H/ACA domain is conserved throughout echinoderm TRs. Comparison of representative TR H/ACA domains from vertebrate, human (A); sea urchin, purple sea urchin (B); brittle star, blunt spined brittle star (C); and feather star, Japanese feather star (D). The namesake box H and ACA moieties (open boxes) are present in echinoderm TRs with the full-length 3′-end identified. The CAB box (open box) is present in sea urchin and brittle star TRs. The CAB box is either lost or cryptic in teleost fish and feather star TRs. Covariation (black bar), universal-invariant between vertebrates and echinoderm (red), and group-invariant (orange) nucleotides for vertebrate, sea urchin, and brittle star TR H/ACA domains is based on multiple sequence alignment of 42 vertebrate (Chen et al. 2000; Podlevsky et al. 2008; Xie et al. 2008), 9 sea urchin/sand dollar, 3 brittle star, and a feather star species (see Supplemental Fig. S5).
	FIGURE 6. The central domain of vertebrate and echinoderm TRs is functionally equivalent yet structurally divergent. Schematic comparison of representative TRs for vertebrate, human (A); sea urchin, purple sea urchin (B); brittle star, blunt spined brittle star (C); and feather star, Japanese feather star (D). Vertebrate and echinoderm TR share structurally homologous template proximal pseudoknot (blue) and H/ACA (orange) domains. The sea urchin and brittle star eCR4/5 (red) are functionally homologous to vertebrate CR4/5 (red). A functionally homologous CR4/5 domain has yet to be determined (red box) for the feather star TR. Numbers below each schematic denote the known and putative (*) size ranges for TRs within each group.

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Autexier, Boundary elements of the Tetrahymena telomerase RNA template and alignment domains. 1995, Pubmed