Logeswaran D et al. (2021), Monophyletic Origin and Divergent Evolution of ...

ECB-ART-49690

Mol Biol Evol 2021 Jan 04;381:215-228. doi: 10.1093/molbev/msaa203.

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Monophyletic Origin and Divergent Evolution of Animal Telomerase RNA.

Logeswaran D , Li Y , Podlevsky JD , Chen JJ .

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Telomerase RNA (TR) is a noncoding RNA essential for the function of telomerase ribonucleoprotein. TRs from vertebrates, fungi, ciliates, and plants exhibit extreme diversity in size, sequence, secondary structure, and biogenesis pathway. However, the evolutionary pathways leading to such unusual diversity among eukaryotic kingdoms remain elusive. Within the metazoan kingdom, the study of TR has been limited to vertebrates and echinoderms. To understand the origin and evolution of TR across the animal kingdom, we employed a phylogeny-guided, structure-based bioinformatics approach to identify 82 novel TRs from eight previously unexplored metazoan phyla, including the basal-branching sponges. Synthetic TRs from two representative species, a hemichordate and a mollusk, reconstitute active telomerase in vitro with their corresponding telomerase reverse transcriptase components, confirming that they are authentic TRs. Comparative analysis shows that three functional domains, template-pseudoknot (T-PK), CR4/5, and box H/ACA, are conserved between vertebrate and the basal metazoan lineages, indicating a monophyletic origin of the animal TRs with a snoRNA-related biogenesis mechanism. Nonetheless, TRs along separate animal lineages evolved with divergent structural elements in the T-PK and CR4/5 domains. For example, TRs from echinoderms and protostomes lack the canonical CR4/5 and have independently evolved functionally equivalent domains with different secondary structures. In the T-PK domain, a P1.1 stem common in most metazoan clades defines the template boundary, which is replaced by a P1-defined boundary in vertebrates. This study provides unprecedented insight into the divergent evolution of detailed TR secondary structures across broad metazoan lineages, revealing ancestral and later-diversified elements.

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	Fig. 1. A phylogeny-assisted approach for TR identification. (A) The workflow of phylogeny-assisted reiterative homology search for novel TRs. An initial PWM and secondary structure pattern derived from known TRs was used to search for novel TR candidates in the genomes or transcriptomes of closely related species. The newly identified TR sequences were used to improve the PWM and structural pattern for searching TR candidates in distantly related species. (B) Comparison of vertebrate and echinoderm TR secondary structures. Two structural domains, T-PK and box H/ACA domains, are conserved in both vertebrate and echinoderm TRs, whereas the vertebrate CR4/5 domain that contains P6.1 stem-loop is replaced with a functionally equivalent eCR4/5 domain in echinoderms. The size ranges of vertebrate and echinoderm TRs are shown. (C) PWMs of TR sequences of the pseudoknot and H/ACA domains. PWMs are derived from the multiple-sequence alignment of 42 vertebrate TRs (Chen et al. 2000) and 13 echinoderm TRs (Li et al. 2013; Podlevsky et al. 2016b). Conserved sequence motifs and base-paired regions within each domain are indicated underneath the matrices
	Fig. 2. Secondary structures of deuterostome TRs. (A) Evolutionary relationship of deuterostome phyla and classes with TR identified. The numbers of TRs identified in this and previous studies for each class are indicated. An asterisk denotes the classes for which the TR secondary structure of a representative species is shown in B–D. Fungi represents the outgroup in the phylogenetic tree. (B–D) Representative TR secondary structures determined by phylogenetic comparative sequence analyses are shown for Asymmetron lucayanum (lancelet) from phylum chordata (B), Saccoglossus kowalevskii (acorn worm) from phylum hemichordata (C), and Acanthaster planci (sea star) from phylum Echinodermata (D). The conserved TR structural domains are shaded in blue. Universal covariations (thick lines), invariant residues, and residues with >80% conservation are based on the sequence alignment of 55 previously identified animal TRs and 82 novel metazoan TRs identified in this study. Group-specific covariations (thick lines) are indicated and based on the sequence alignment of TRs from individual groups including 50 chordate TRs (42 previously identified and eight novel), 23 echinoderm (13 previously identified and ten novel), and four acorn worm TRs.
	Fig. 3. Functional validation and characterization of acorn worm TR. (A) Direct telomerase activity assay of acorn worm telomerase reconstituted in vitro. (top) Template sequence of acorn worm TR (open box) with base-pairing of six permuted telomeric DNA primers a–f. Sequence and number of expected nucleotides added are depicted for each primer. (bottom) Direct primer-extension assay of acorn worm telomerase. Acorn worm telomerase was in vitro reconstituted from T7 transcribed SkoTR (436 nt) and SkoTERT synthesized in RRL. The reconstituted acorn worm telomerase was analyzed with six permuted telomeric DNA primers (lanes 2–7). A reaction omitting SkoTR was included as a negative control (lane 1). A 32P end-labeled 18-mer oligonucleotide was added to each reaction as recovery control (r.c.) prior to ethanol precipitation of DNA products. Numbers to the right of the gel indicate the number of repeats or nucleotides added to the primer. (B) Two essential fragments of SkoTR. The T-PK and CR4/5 fragments of SkoTR were synthesized separately and assembled with SkoTERT in RRL, followed by telomerase activity assay. The schematic secondary structures of the SkoTR T-PK (top) and CR4/5 (bottom) fragments. Nucleotide numbers denote the 5′- and 3′-ends of the T7 transcribed RNA fragments, T-PK and CR4/5. Positions of two highly conserved U residues (U289 and U290) in the P6.1 loop of CR4/5 domain are shown. (C) Minimal requirement of TR domains for telomerase activity. T7 transcribed SkoTR fragments, T-PK (nt 1–184) and CR4/5 (nt 237–307), were assembled with in vitro synthesized SkoTERT and analyzed for activity. The CR4/5 fragments with a P6.1 substitution (U289C or U290C) were assembled with T-PK fragment and SkoTERT and assayed for activity with the primer (GGGTTA)3. A 32P end-labeled 18-mer oligonucleotide was added to each reaction prior to ethanol precipitation of DNA products as r.c. The numbers of repeats added to the primer are shown to the right of the gel.
	Fig. 4. Secondary structures of protostome TRs. (A) Evolutionary relationship of protostome phyla and classes with TR identified. The numbers of TRs identified in this study for each class are indicated. An asterisk denotes the classes for which the TR secondary structure of a representative species is shown in B–D. Fungi represents the outgroup in the phylogenetic tree. (B–D) Representative TR secondary structures determined by phylogenetic comparative sequence analyses are shown; Pomacea diffusa (apple snail) from phylum mollusca (B), Eisenia fetida (earth worm) from phylum annelida (C), and Lingula anatina (tongue shell) from phylum brachipoda (D). The conserved TR structural domains are shaded in blue. The eCR4/5 domain is shaded in red. Universal covariations (thick lines), invariant residues, and residues with >80% conservation are based on the sequence alignment of 55 previously identified animal TRs and 82 novel metazoan TRs identified in this study. Group-specific covariations (thick lines) are indicated and based on the sequence alignment of TRs from individual groups including 21 mollusca, 7 annelida, 1 brachiopda, and 1 phoronida TRs, respectively.
	Fig. 5. Functional validation and characterization of apple snail TR. (A) Direct telomerase activity assay of acorn worm telomerase reconstituted in vitro. (top) Template sequence of acorn worm TR (open box) with base-pairing of six permuted telomeric DNA primers a–f. Sequence and number of expected nucleotides added are depicted for each primer. (bottom) Direct primer-extension assay of apple snail telomerase. Apple snail telomerase was in vitro reconstituted from T7 transcribed PdiTR (408 nt) and PdiTERT synthesized in RRL. The reconstituted acorn worm telomerase was analyzed with six permuted telomeric DNA primers (lanes 1–6). A 32P end-labeled 18-mer oligonucleotide was added to each reaction as recovery control (r.c.) prior to ethanol precipitation of DNA products. Numbers to the right of the gel indicate the number of repeats or nucleotides added to the primer. (B) Two essential fragments of PdiTR. The T-PK and CR4/5 fragments of PdiTR were synthesized separately and assembled with PdiTERT in RRL, followed by telomerase activity assay. The schematic secondary structures of the PdiTR T-PK (top) and eCR4/5 (bottom) fragments. The eCR4/5 domain consists of three stems, P4–P6. Nucleotide numbers denote the 5′- and 3′-ends of the T7 transcribed TR fragments, T-PK, P4/5/6, P5, and P6. (C) Minimal requirement of TR domains for telomerase activity. T7 transcribed PdiTR fragments, T-PK (nt 1–146), P4/5/6 (nt 162–335), P5 (nt 191–244), or P6 (nt 245–309), were assembled with in vitro synthesized PdiTERT and analyzed for activity using the primer (GGGTTA)3. The PdiTR fragments included in each reaction are indicated above the gel. A 32P end-labeled 18-mer oligonucleotide was added to each reaction prior to ethanol precipitation of DNA products as r.c. The number of nucleotides added to the primer is shown to the right of the gel. (D) The effect of P1.1 position on template boundary definition. Two PdiTR mutants, L1 and L2, with a single adenosine residue inserted immediately after positions 11 and 32, respectively, were assembled in vitro with PdiTERT and assayed for telomerase activity using primer (GGGTTA)3.
	Fig. 6. Secondary structures of basal metazoan TRs. (A) Evolutionary relationship of basal metazoan phyla and classes with TR identified. The numbers of TRs identified in this study for each class are indicated. An asterisk denotes the classes for which the TR secondary structure of a representative species is shown in B–D. Fungi represents the outgroup in the phylogenetic tree. (B–D) Representative TR secondary structures determined by phylogenetic comparative sequence analyses are shown for Acropora digitifera (digitate coral) from phylum cnidaria (B), Trichoplax adhaerens (flat animal) from phylum placozoa (C), and Amphimedon queenslandica (common sponge) from phylum porifera (D). The conserved TR structural domains are shaded. Universal covariations (thick lines), invariant residues, and residues with >80% conservation are based on the sequence alignment of 55 previously identified animal TRs and 82 novel metazoan TRs identified in this study. Group-specific covariations are indicated and based on the sequence alignment of TRs from individual groups including 26 cnidaria, 1 placozoa, and 3 porifera species.
	Fig. 7. Monophyletic origin and divergent evolution of TR structural elements and biogenesis pathways. Simplified phylogenetic trees of major metazoan (A) and eukaryotic (B) lineages shown. Branch lengths do not represent evolutionary distance. (A) Evolution of TR structural elements across metazoan lineages. Loss of RNA secondary structural elements P1.1, P2.1, or P6.1 is indicated in oval. Gain of P2a or P2a.1 is shown in rectangle. Specific events of loss and gain of TR structural elements are marked along the respective metazoan lineages. (B) Divergence of TR biogenesis pathway and transcription machinery across eukaryotes. The TR transcription machinery in each eukaryotic kingdom is indicated as Pol II or Pol III. The size range of TRs from each group is indicated. Biogenesis pathway of respective eukaryotic clades are shown to the right.

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