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A Widespread Class of Reverse Transcriptase-Related Cellular Genes

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A Widespread Class of Reverse Transcriptase-Related Cellular Genes FEATURE A widespread class of reverse transcriptase-related SACKLER SPECIAL cellular genes Eugene A. Gladyshev1 and Irina R. Arkhipova2 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA 02543 Edited by Marlene Belfort, Wadsworth Center, Albany, NY, and approved August 2, 2011 (received for review March 3, 2011) Reverse transcriptases (RTs) polymerize DNA on RNA templates. function. In this study, we identify and characterize the second They fall into several structurally related but distinct classes and major group of RT-related cellular genes. form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) syn- Results thesizing RNA on RNA templates. It is generally believed that most Identification and Characterization of rvt Genes in Bdelloid Rotifers. RT-like enzymes originate from retrotransposons or viruses and We discovered rvt genes in the course of cloning and sequencing have no specific function in the host cell, with telomerases being of telomeric regions from rotifers of the class Bdelloidea, small the only notable exception. Here we report on the discovery and freshwater invertebrates that are best known for having evolved for properties of a unique class of RT-related cellular genes collectively tens of millions of years apparently without males and meiosis, for named rvt. We present evidence that rvts are not components of their resistance to desiccation and ionizing radiation, and for their retrotransposons or viruses, but single-copy genes with a character- ability to acquire foreign genes from diverse sources (11, 13–16). istic domain structure that may contain introns in evolutionarily During sequencing of fosmids from the genomic library of the conserved positions, occur in syntenic regions, and evolve under bdelloid rotifer Adineta vaga (family Adinetidae), we found a purifying selection. These genes can be found in all major taxonomic member of a previously unrecognized group of RT-like genes, groups including protists, fungi, animals, plants, and even bacteria, which we named rvt because it contains an identifiable rvt con- although they exhibit patchy phylogenetic distribution in each king- served domain (reverse transcriptase; pfam00078:RVT_1). The dom. We also show that the RVT protein purified from one of its A. vaga rvt did not fall into any of the known RT categories, such GENETICS natural hosts, Neurospora crassa, exists in a multimeric form and has as retrons, retroplasmids, group II introns, telomerases, non-LTR the ability to polymerize NTPs as well as dNTPs in vitro, with a strong retrotransposons, LTR retrotransposons, retroviruses, and para- preference for NTPs, using Mn2+ as a cofactor. The existence of a pre- retroviruses. Its single-copy status was established by Southern blot viously unknown class of single-copy RT-related genes calls for hybridization of genomic DNA and by exhaustive screening of the reevaluation of the current views on evolution and functional roles A. vaga genomic library (17). In this library, we found a colinear of RNA-dependent polymerases in living cells. pair of rvt-containing fosmids with 4% overall divergence, consis- tent with the genome structure of bdelloid rotifers in which NA-dependent polymerases are essential for cellular function, chromosomes occur as colinear allelic pairs with overall di- as they mediate the flow of genetic information from DNA to vergence up to 6% (17, 18). The divergence between members of D < RNA to proteins (1). In contrast, RNA-dependent polymerases the rvt pair is 1%, either at the nucleotide level (13/2,490 have long been associated with replication of selfish and parasitic nt substitutions) or at the protein level (6/829 aa substitutions). genetic elements, such as viruses or transposons. Although the Sequencing of rvt-containing fosmids revealed that rvt genes are discovery of reverse transcriptase (RT) challenged the concept of located in a subtelomeric region rich in telomeric repeats, telo- unidirectionality of the flow of genetic information, this reverse mere-associated Athena retrotransposons, and foreign genes of direction has been reserved for retroviruses, pararetroviruses apparently bacterial or fungal origin (Fig. S1A). rvt (hepadna- and caulimoviruses), and other RT-containing multi- Moreover, we found in four other species of bdelloid rotifers (Philodina roseola, Philodina acuticornis,andMacrotrachela copy entities such as non-LTR and LTR retrotransposons, group II quadricornifera from the family Philodinidae and Habrotrocha rosa introns, retrons, and retroplasmids, as well as occasional retro from the family Habrotrochidae), using PCR and genomic library (pseudo)genes (2–4). Similarly, viral RNA-dependent RNA poly- screens. On a contig from the P. roseola genomic library, rvt is merases (RdRPs), enzymes structurally related to RTs, serve to located between two genes of apparently bacterial origin (Fig. replicate the genomes of viruses that use RNA as genetic material S1B). There are two distinct lineages of rvt genes in bdelloids, A (5). These and certain other polymerases are unified by the archi- tecture known as “right hand,” composed of the three subdomains called fingers, palm, and thumb (6). Like all polymerases, they use This paper results from the Arthur M. Sackler Colloquium of the National Academy of two-metal-ion catalysis for phosphoryl transfer reactions resulting Sciences, “Telomerase and Retrotransposons: Reverse Transcriptases that Shaped Ge- in nucleotide addition. nomes” held September 29–30, 2010, at the Arnold and Mabel Beckman Center of the In 1997, this diverse superfamily of enzymes was joined by the National Academies of Sciences and Engineering in Irvine, CA. The complete program and telomerase reverse transcriptase (TERT), a specialized RT that audio files of most presentations are available on the NAS Web site at www.nasonline. org/telomerase_and_retrotransposons. maintains the ends of eukaryotic linear chromosomes by addition Author contributions: E.A.G. and I.R.A. designed research; E.A.G. and I.R.A. performed of short G-rich repeated DNA sequences that are copied multiple research; E.A.G. contributed new reagents/analytic tools; E.A.G. and I.R.A. analyzed data; times via reverse transcription of a specific region of the associated and I.R.A. wrote the paper. RNA template constituting part of the holoenzyme (7). Telo- The authors declare no conflict of interest. merases are unique in being single-copy eukaryotic RT genes that This article is a PNAS Direct Submission. do not represent a component of any mobile element or virus. It Data deposition: The sequences reported in this paper have been deposited in the Gen- has been argued that, in early eukaryotic evolution, telomerases Bank database (accession nos. JN235987–JN235989). may have either descended from domesticated retrotransposons or 1Present address: Department of Molecular and Cellular Biology, Harvard University, given rise to them (8, 9). In fact, TERTs were shown to be most Cambridge, MA 02138. closely related to RTs from Penelope-like retroelements (PLEs) 2To whom correspondence should be addressed. E-mail: [email protected]. – (10 12). However, TERT genes so far remain the only example of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. single-copy RT-related eukaryotic genes with a defined cellular 1073/pnas.1100266108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1100266108 PNAS | December 20, 2011 | vol. 108 | no. 51 | 20311e20316 Downloaded by guest on September 26, 2021 and B, which could originate from two independent acquisition These genes are present in a highly diverse set of species with se- events (Fig. S1C). However, although the A. vaga rvtA has a slightly quenced genomes: 60 fungi (not only euascomycetes and basidio- higher GC content than neighboring genes, there is no detectable mycetes, but also chytrids and microsporidia, the most basal fungal difference in GC content and codon usage between P. roseola rvtB taxa); the moss Physcomitrella patens; six stramenopiles (hetero- and adjacent genes, indicating that if it was also acquired by lateral konts), including the genera Phytophthora, Saprolegnia,and transfer, it took place a sufficiently long time ago for the differ- Pythium; and a bacterium (Fig. 2 and Table S1). In EST data- ences to have ameliorated. bases, there are also two homologous fragments from an ar- thropod (Arctic springtail Onychiurus arcticus), which, however, Structure and Distribution of rvt in Sequenced Genomes. Compari- exhibit some similarity to rvt from a microsporidian parasitizing son of rotifer rvt genes with their homologs in other kingdoms on mosquitoes (Vavraia culicis). Of special interest is the exis- reveals their highly conserved overall structure, which deviates tence of rvt genes in the sequenced genome of the filamentous significantly from all presently known RT types (Fig. 1). A typical gliding bacterium Herpetosiphon aurantiacus (Chloroflexi) and rvt ORF is 800–1,000 aa in length. The core RT domain, which two uncultured environmental bacteria. Finding the same type of contains RT motifs 1 through E (fingers and palm), plus the thumb RT in both prokaryotes and eukaryotes is so far unprecedented. subdomain, is framed at the N and C termini by well-conserved However, despite its basal position, this RT may have originated 300- and 200-aa extensions, respectively, which have no homology from a rare
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