Telomere-associated endonuclease-deficient Penelope-like retroelements in diverse eukaryotes

Abstract

The evolutionary origin of telomerases, enzymes that maintain the ends of linear chromosomes in most eukaryotes, is a subject of debate. Penelope-like elements (PLEs) are a recently described class of eukaryotic retroelements characterized by a GIY-YIG endonuclease domain and by a reverse transcriptase domain with similarity to telomerases and group II introns. Here we report that a subset of PLEs found in bdelloid rotifers, basidiomycete fungi, stramenopiles, and plants, representing four different eukaryotic kingdoms, lack the endonuclease domain and are located at telomeres. The 5' truncated ends of these elements are telomere-oriented and typically capped by species-specific telomeric repeats. Most of them also carry several shorter stretches of telomeric repeats at or near their 3' ends, which could facilitate utilization of the telomeric G-rich 3' overhangs to prime reverse transcription. Many of these telomere-associated PLEs occupy a basal phylogenetic position close to the point of divergence from the telomerase-PLE common ancestor and may descend from the missing link between early eukaryotic retroelements and present-day telomerases.

Fig. 1.

Flow chart of the chromosome end enrichment procedure (see Materials and Methods for details).

Fig. 2.

Structural organization of telomere-associated retroelements. Each red letter T indicates point of PLE 5′ truncation and addition of reverse-complement telomeric repeats at a chromosome end; 5′ truncation points within individual copies are shown by thin diagonal lines; reverse-complement telomeric repeat units are specified for each species. Noncoding sequences are shown by a thin line; PLE ORFs are shown by an open rectangle with the N- and C-terminal domains (N, C) and the central region, which includes the seven core RT motifs (RT1–RT7) and the thumb domain (TH). J, 5′ truncation point in an upstream copy when joined to a full-length downstream copy, forming a “pseudoLTR” (see also SI Fig. 7); O, point of addition of Athena-AvO to -AvN at the O1, O3, and N1 telomeres containing both elements in the same orientation. Small red boxes mark the position of short internal telomeric repeat stretches; larger boxes mark longer tandem repeats (shown in SI Fig. 7); introns are denoted by triangles. Telomeric minilibrary clones from telomeres M1–M2, O1–O3, and N1–N2 in A. vaga and C, K in P. roseola (also listed in SI Table 1) are aligned with the corresponding Athena sequences. Also shown is the position of Athena-specific primers used for RT-PCR (black, paired), STELA (orange), and 5′ RACE (purple) (see Fig. 3 b–d for experiments). Only Athena variants found at telomeres are shown; additional diverged variants were identified on sequenced cosmids/fosmids but have not yet been found at telomeres and are not presented here. ◆, nuclear localization signals; cccc, coiled-coil domains; LZ, leucine zipper motif. (Scale bar, 1 kb.).

Fig. 3.

Characterization of bdelloid Athena elements. (a) Structure of telomeres M1, O3, and O4 in Athena-containing fosmids obtained from the A. vaga genomic library. Color codes and ORFs are as in Fig. 2; telomeres are in red; truncated Athena copies are delimited with ∼ (vertical or horizontal). There are 10 and 8 48-bp repeats between AvO and AvN in the O3 and O4 telomeres, respectively. Juno1.4 is a slightly 3′ truncated copy of an LTR retrotransposon in an inverse orientation (41). (Scale bar, 1 kb.) (b) STELA. The rationale (12) is shown on the top. A telorette oligo is annealed to the G-rich overhang and, after ligation, a specific telomere is amplified with the teltail primer and the primer in the subtelomeric region. The EtBr-stained gel shows amplification of telomeres M and O with the corresponding Athena primers (Fig. 2a; Materials and Methods); below is the same gel probed with (TGAGGG)₄ for visualization of telomeric repeat-containing amplicons. As a control, lanes marked (Telorette−) contained no telorette oligos in the ligation mix. Amplification of telomeres M1, O1, and O3 was confirmed by cloning and sequencing of total PCR products. (c) 5′ RACE for AvN and AvO. Arrows indicate the position of RNA start sites relative to ORF1, obtained by sequencing of the corresponding amplicons. The level of AvM transcription (d) was insufficient to generate a RACE product. (d) RT-PCR of A. vaga poly(A)⁺ RNA with AvM, AvO, and AvN primers (see Fig. 2a). All upper bands correspond in sequence to the unspliced message; lower bands are spliced at the predicted intron boundaries (AvN) or result from cryptic splicing (AvO).

Fig. 4.

Bootstrap network of 46 PLE and TERT sequences based on maximum-likelihood (ML) distances estimated with a WAG substitution matrix plus an eight-category gamma rate heterogeneity correction. The data set included 700 characters from the core RT and its N-terminal and C-terminal extensions (SI Fig. 6). A 370-aa RT fragment of an early branching PLE was found in the slime mold, Physarum polycephalum (Amoebozoa), but no evidence is yet available for its association with telomeres because of insufficient genome coverage. This PLE contains an insertion between motifs RT3 and RT4 called IFD (insertion into the fingers domain), which is found only in TERTs and is important for TERT function, apparently stabilizing very short DNA-RNA hybrids (42). EN(−) retroelements shown in Fig. 2 (AvM, AvO, AvN, PrR, Cc1, Pc1, Pc2, Pt1, Sm1, and Sm2) are underlined; EN(+) indicates the presence of EN domain in Neptune and Poseidon/Penelope groups (full element and species names are given in SI Fig. 6). The Coprina group may or may not be monophyletic. Triangle indicates the midpoint. For clade support values, see SI Fig. 9b.

Fig. 5.

Model for EN-independent terminal retrotransposition. Red, retroelement sequences; blue, chromosomal DNA; pale ovals, proteins that normally form caps at the telomeres. Priming at the G-rich 3′ overhang is facilitated by annealing with reverse-complement telomeric repeats in the 3′ UTR of the RNA template. In the absence of telomeric repeats, annealing at microhomologies could be assisted by ORF1. Telomeric repeats are added by telomerase, after which the normal capping structure is restored. Note that the second-strand synthesis would not require special mechanisms other than routine DNA replication as occurs during C-rich strand synthesis (not to scale).