and : Reverse transcriptases that shaped genomes

Marlene Belforta,b,c,1, M. Joan Curciob,c,1, and Neal F. Lued,1

aDepartment of Biological Sciences and RNA Institute, University at Albany, State University of New York, Albany, NY 12222; bWadsworth Center, New York State Department of Health, Albany, NY 12201-2002; cDepartment of Biomedical Sciences, University at Albany, State University of New York, Albany, NY 12222; and dDepartment of Microbiology and Immunology, W. R. Hearst Microbiology Research Center, Weill Medical College of Cornell University, New York, NY 10065

“Two independent groups of investigators while maintaining cellular balance and then the cDNA is inserted into the ge- have found evidence of an ... which genome integrity. nome via integrase (5). synthesizes DNA from an RNA template. The colloquium addressed the evolution This discovery, if upheld, will have impor- and interrelationships of RTs of viruses, Evolutionary Relationships tant implications... information transfer ” transposons, telomeres, and cellular genes, It has been appreciated for some time that from DNA to RNA can be inverted. 1970, telomerase and RTs ex- preamble to refs. 1 and 2. while exploring RT structure, function, and its bearing on development, aging, and hibit a number of structural and mecha- hese words accompanied two disease. It remains debatable whether nistic similarities and are likely to share articles describing the discovery represent domesticated ret- a common origin (6–11). Phylogenetic Tof RNA-dependent DNA poly- rotransposons or whether these chromo- analyses reinforce a deep-rooted connec- merase, now known as reverse some-capping agents radiated into mobile tion between these two families of poly- transcriptase (RT), in the virions of RNA genetic elements. Nevertheless, their phy- merases (Fig. 1C). Irina Arkhipova’s tumor viruses. RT activity was recognized logenetic and functional relatedness is lecture, as well as the accompanying arti- independently by David Baltimore (1) and clear (Fig. 1), and indeed, retrotrans- cle on a distinct group of RTs (12), ex- Howard Temin and Satoshi Mizutani (2). posons can provide alternative means of plored evolutionary space between the In five printed pages these authors col- maintaining chromosome ends, as will large class of RTs that are associated with fi lectively challenged the unidirectionality become evident from the following. diverse sel sh mobile retroelements and “ of macromolecular synthesis, from DNA The colloquium and this compendium of telomerase RT. She described an evolu- ” to RNA to , the central dogma of articles reflect the lifestyle of , tionary intermediate, a unique family of molecular biology. Indeed by demonstrat- retrotransposons, and telomeres. The single-copy RT genes that is not only dis- ing that DNA synthesis can be templated retrotransposons we consider are the tinct from the telomerase RT gene but by RNA, they overturned the central retroviral-like elements that contain long also shows none of the hallmarks of mo- dogma. However, there was no way of terminal repeats [LTR retrotransposons bile element association. This RT gene predicting how in the ensuing 4 decades (e.g., Ty elements)] and those that do not family seems to have ancient origins and is found sporadically throughout all eukary- scientists would uncover the essential role contain LTRs [e.g., long interspersed otic kingdoms. Thus, telomerase is not the of RT in shaping genomes and making nuclear elements (LINE) elements and only single-copy gene that evolved from possible the diversity of life on earth. group II introns]. These non-LTR retro- an ancestral RT gene. This finding lends This was the topic of the colloquium transposons are also referred to as target- credence to the idea that different classes “Telomerase and Retrotransposons: Re- primed (TP) elements, for their mecha- of retroelements evolved independently verse Transcriptases that Shaped Ge- nism of chromosome integration (3). The from divergent single-copy RT genes. nomes,” held September 29–30, 2010, architecture of the different retroelements Another connection between telomeres which forms the basis of this special fea- under consideration is schematized in Fig. and retrotransposons was provided ture. We now know that approximately 1A, indicating that they are all molecular fi by Mary-Lou Pardue, who described half of the human genome and a signi - mosaics, with different functional modules Drosophila telomeres, which are main- cant portion of every other eukaryotic associated with RT. RT is fused to pro- tained not by telomerase but rather by re- genome is generated by RTs, which are tease and integrase domains in both ret- peated retrotransposition of a small set of present in two major forms: telomerases, roviruses and LTR retrotransposons, retroelements HeT-A, TART, and TAHRE which synthesize the ends of linear eu- whereas RT is juxtaposed to endonuclease (13). In a “natural” experiment in the karyotic chromosomes, and retrotrans- domains in TP retrotransposons and some poson RTs, which deposit copies of group II introns. Retrotransposition of themselves throughout the genetic land- a TP retrotransposon or group II intron is This paper results from the Arthur M. Sackler Colloquium scape and generate pseudogenes with usually initiated by endonucleolytic cleav- of the National Academy of Sciences, “Telomerase and spectacularly diverse roles in gene regula- age, exposing a 3′-OH that serves as the Retrotransposons: Reverse Transcriptases that Shaped Ge- nomes” held September 29–30, 2010, at the Arnold and tion. There was no inkling 40 years ago primer for reverse , with the Mabel Beckman Center of the National Academies of Sci- that misregulation of RTs can cause RNA of the element being the template ences and Engineering in Irvine, CA. The complete program chromosomal catastrophes that are char- (Fig. 1B). This process of target-primed and audio files of most presentations are available on the acteristic of cancer, neurological disorders, reverse transcription (TPRT) (4) is also NAS Web site at trotransposons. and aging, as occurs when telomerase and used for cDNA synthesis at telomeres, Author contributions: M.B., M.J.C., and N.F.L. wrote the retrotransposon RTs escape normal con- where the telomerase RT (TERT) uses paper. trols. A central question in genome bi- a3′-OH that occurs naturally at chromo- The authors declare no conflict of interest. ology then is how the activity of RTs has some ends to prime TPRT. In contrast, 1 fi To whom correspondence may be addressed. E-mail: been harnessed, regulated, and modi ed LTR retrotransposons and retroviral RNA [email protected], [email protected], or nflue@ throughout evolution to generate diversity templates are first reverse transcribed, and

20304–20310 | PNAS | December 20, 2011 | vol. 108 | no. 51 Downloaded by guest on September 27, 2021 AKE PCA ETR:INTRODUCTION FEATURE: SPECIAL SACKLER

Arabidopsis thaliana, one of the two TERs acts as a repressor rather than a stimulator of telomere elongation, possibly by form- ing an inactive complex with the catalytic TERT (15). This finding illustrates the adaptability of telomerase components. The versatility of the telomerase ribo- nucleoprotein (RNP) is further reflected in the diversity of DNA sequences that it synthesizes. Neal Lue highlighted the ex- traordinary variability of the telomere re- peat units in a group of budding yeasts that include both Saccharomyces and Candida species. Whereas the repeat units are 5–8 bp and G-rich in most phyla, the ones in budding yeast can be as long as 25 bp and exhibit little nucleotide bias on either strand (16). The selection pressures that resulted in telomere sequence divergence in these and other organisms are not understood and are worthy of further analyses. Another example of the plasticity of retroelements is the group II intron, which is thought to be of bacterial origin and is widely hypothesized to be ancestral to the prevalent eukaryotic spliceosomal introns. Group II intron RNAs are unusual retro- elements in that they are ribozymes that encode RT. After self-splicing, the intron can reverse-splice into a DNA target and be reverse transcribed by TPRT, similar to TP-retrotransposition and telomere syn- ′– ′ Fig. 1. Featured retroelements. (A) Architecture of retroelements. RNA maps (5 3 , green) show RT thesis (3, 17). Marlene Belfort’s lecture (red). The schematic is not to scale. Stem-loops flanking the group II intron RT represent catalytic RNA, and the wavy line represents the RNA component of telomerase (TER). The hatches indicate that TERT addressed the paradox that arises from the and TER are encoded by separated genes. Boxed triangles designate LTRs. Coding sequences are GAG, compelling hypothesis that group II in- Gag protein; PR, protease; IN, integrase; , envelop protein; EN, endonuclease; TEN, telomerase es- trons, found exclusively in prokaryotes and sential N terminus; and TRBD, telomerase RNA-binding domain. (B) Initiation of cDNA synthesis by RT. eukaryotic organelles, are the progenitors RTs are shown at telomere and within chromosome. TERT acts on 3′-OH at a telomere, whereas the RT of of spliceosomal introns, which are unique EN-proficient TP retrotransposons acts on the 3′-OH at a double-strand break. The green wavy line in- to and abundant in eukaryotic nuclei. To dicates RNA. (C) Phylogram of RT classes. Phylogram shows clustering of RT members, as described in understand why a eukaryotic nucleus is detail in the article by Gladyshev and Arkhipova (12). (D) Schematic of telomerase. Telomerase RNA (TER, inhospitable to group II introns even green) with its pseudoknot structure has the template region base-paired to telomere DNA (black) in the though their apparent descendants are active site of telomerase RT (TERT, red). The diagram is not to scale. ubiquitous, the Belfort group introduced a group II intron into nuclear genes (18). genus Drosophila, and perhaps other in- organisms results in the activation of re- The group II intron was spliced; however, sects, the loss of telomerase apparently led combination pathways at telomeres. These splicing was primarily cytoplasmic, and for reasons that remain enigmatic, the spliced to the utilization of other RTs that have pathways have been extensively charac- transcript was not translated. Perhaps coevolved with their host cells to provide terized in yeast but not in mammalian cells gene compartmentalization in nuclei, ex- a robust mechanism for maintaining (14). Tammy Morrish (Greider labora- clusion of group II introns, and biogenesis HeT-A chromosome ends. Although , tory) described her ongoing study of the of spliceosomal introns were all driven by TART, and TAHRE resemble typical non- genetic requirement for telomere re- the need to protect genes from the muta- LTR retrotransposons, they have several combination using tumor and immortal- genic effects of group II retroelements. distinctive features contributing to their ized cells derived from telomerase effectiveness at the telomere. Further ev- knockout mice. Her preliminary data point Telomerases and Retrotransposons idence of collaboration between the host to the involvement of the recombination Function Within the Context of Highly and these elements is seen in the se- function of RAD50. An intriguing and Structured RNPs quence of a fragment of a telomere array, unresolved question is whether in this A detailed understanding of telomerase moved into a centromere region by an setting retrotransposons could also play mechanisms will ultimately require high- ancient transposition and later remodeled a role in capping the chromosome ends. resolution structural information. Impres- to resemble the chromatin of other cen- Beyond the recurring questions con- sive progress toward achieving this goal was tromere regions—chromatin that is very cerning the telomerase–retrotransposon made by the determination of the atomic different from that of the analogous se- relationship, unusual aspects of telomer- resolution structure of TERT from Tribo- quence remaining at telomeres. ase and telomere evolution are emerging. lium castaneum (19). At the colloquium However, this rather astonishing feat of Dorothy Shippen presented a case of te- and in an accompanying perspective, Juli substitution does not seem to occur fre- lomerase RNA (TER) duplication and Feigon summarized advances on the quently, because loss of telomerase in most functional specialization. In the plant structure of human telomerase RNA

Belfort et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20305 Downloaded by guest on September 27, 2021 (TER) and the RNP (20, 21). The non- paction is required to achieve the catalyt- forms a virus-like particle (VLP) wherein coding TER not only supplies the tem- ically active state. the RNA is reverse transcribed. In her plate for reverse transcription but also presentation, Suzanne Sandmeyer pro- stimulates the polymerization by TERT Tricks to Accomplish the Complex Task posed that partitioning of the Ty3 LTR through mechanisms that remain poorly of Reverse Transcription retrotransposon occurs when translating understood (Fig. 1D). Two conserved A unique property of telomerase as an RT Ty3 RNA interacts with mRNA trans- TER structural elements seem to be par- is its ability to make consecutive copies of lation suppressors and decay that ticularly important for the stimulatory telomeric repeats using a short template concentrate in stress granules and mRNA function: a template/pseudoknot domain (Fig. 1D), referred to as repeat addition processing bodies. Indeed, shortly after (the core domain) and a stem-terminus processivity. To accomplish this task, te- induction of Ty3 expression, Ty3 Gag and element (STE) (22). Structural models of lomerase must undergo a translocation RNA colocalize in microscopically distinct subdomains of these elements have been reaction, as follows: after each round of cytoplasmic foci that resemble stress determined in recent years, primarily by reverse transcription, the RNA–DNA hy- granules and processing bodies, and con- NMR. Feigon presented a structure model brid dissociates and TERT realigns the 3′ tain many of the same sequestration and of the entire core domain based on struc- end of the DNA to the RNA template, decay factors, but are formed under sep- tures and dynamics of all of the helical via a putative anchor site in TERT, to arate circumstances. Sandmeyer has subdomains, which revealed an intriguing enable the next round of polymerization. named these foci “retrosomes,” reflecting V-shaped conformation. Additionally, on Insights into these molecular gymnastics the observation that components of other the basis of the structure of the Tribolium were provided by two presentations. Kathy retrotransposons, including the human L1 castaneum TERT, she proposed two Collins described her investigation of the element and eukaryotic retroviruses, lo- plausible views of the relative disposition human telomerase core complex through calize to similar cytoplasmic bodies. It is of the core domain and TERT. A parallel coexpression of truncated TERT protein likely that sequestration of Ty3 RNA with advance on the STE is reported by Julian and TER RNA domains, as well as anal- stress granule/processing body proteins is Chen and colleagues in an accompanying ysis of the resulting RNP. Particularly mediated by the VLP structural protein, article (23). Using a unique photoaffinity noteworthy was her finding that the Gag, because neither Ty3 RNA nor Gag cross-linking approach and mass spec- telomerase essential N-terminal (TEN) proteins localize to retrosomes if Gag trometry, they identified three nucleotide– domain of TERT is dispensable for binding to Ty3 RNA is blocked (28). amino acid contacts between a vertebrate nucleotide addition but can complement Retrosomes may function to concentrate STE (known as CR4/5) and the corre- the rest of the complex in trans to re- Ty3 VLP components to ensure efficient sponding TERT, thus bringing into constitute a highly processive telomerase VLP assembly. sharper focus the architecture of the (26). This finding reinforces the long- The RNA genomes of LTR retro- telomerase RNP. standing hypothesis that the TEN domain, transposons and retroviruses are reverse Another powerful approach for eluci- which is known to have a weak single- transcribed within RNP complexes in the dating the assembly and mechanisms of stranded DNA-binding activity, may serve cytoplasm, and then the cDNA is in- RNPs is single-molecule FRET, as illus- as the anchor site of telomerase that en- tegrated into the host genome. Host pro- trated in an accompanying article by ables the RNP to trap telomeric DNA teins transcribe the retrotransposon or Xiaowei Zhuang (24). By labeling specific during repeated cycles of extension and integrated . This generates another residues within the Tetrahymena telomer- RNA–DNA dissociation. Julian Chen ad- round of template RNAs for protein syn- ase RNA with fluorescent donors and ac- dressed the fate of the RNA–DNA hybrid thesis. The RNAs can also be used to ceptors, Zhaung and coworkers were able using a clever “template-free” assay, in generate additional genomes for VLPs to monitor the conformation of a pseu- which the template region of the RNA was (LTR retrotransposons) or viral particles doknot in the enzyme complex. They selectively removed from the enzyme (retroviruses). This highly complex mode showed that the TERT protein prevents preparation and supplied in trans as a part of viral particle replication requires that the misfolding of RNA and that only of an RNA–DNA hybrid substrate. By reverse transcription is coordinated with RNPs with properly formed pseudoknots analyzing the binding properties and ac- other obligatory steps during cellular rep- are catalytically active. These observations tivities of this reconfigured telomerase and lication. Insights into this regulation was provide compelling support for the im- a series of mutants, he concluded that provided by Xiaowei Zhuang, who de- portance of a correctly folded pseudoknot a key determinant of translocation effi- scribed elegant single-molecule experi- in telomerase activity, a recurrent idea ciency is the ability of the telomerase ac- ments in which FRET was applied to that has lacked decisive confirmation. tive site to reengage the RNA–DNA monitor the dynamics of individual HIV-1 Although knowledge of the structure of hybrid (27). This proposition in turn sug- RT initiation complexes (29). Her studies retrotransposon RNPs is less advanced gests that the active site is temporarily reveal a dynamic interaction between than that of telomerase, the group II intron disengaged from the RNA template region HIV-1 RT and viral RNA:tRNA template- RNP is beginning to yield to biophysical during translocation and that the telo- primer during initiation of reverse tran- analysis. By purifying the intron RNA in merase RNP is even more dynamic than scription. While paused in the initiation complex with the intron-encoded protein previously envisioned. phase, RT flips between two opposite from its native Lactococcal host, the Bel- A key difference between retro- orientations: one is competent for exten- fort group in collaboration with the Joa- transposons and telomerase is that retro- sion of the primer, whereas the other is chim Frank (Columbia University) and transposon RTs are encoded by the RNA not. Once a stem-loop structure located Greg Van Duyne (University of Pennsyl- template used for reverse transcription, upstream of the primer-binding site in the vania) laboratories has obtained a glimpse whereas telomerase RT and RNA are HIV-1 RNA is traversed, only the orien- of an active RNP. Cryo-EM, size-exclusion encoded by separate genes (Fig. 1A). Thus, tation that places the active site of RT at chromatography, and sedimentation anal- retrotransposons face the problem of the site of primer extension is favored, and yses revealed the intron RNP precursor as partitioning their RNA genomes between elongation is rapid. Zhuang speculated a large, loosely packed structure, in con- translation and reverse transcription. The that pausing at the stem-loop trast to the compact spliced intron RNP RNA of LTR retrotransposons is used as may serve to stall reverse transcription (25). These results suggest that a major a template for synthesis of not only RT but until the virion has adopted its mature conformational change and RNP com- also of the structural protein Gag, which form and enters a new cell, whereupon the

20306 | Belfort et al. Downloaded by guest on September 27, 2021 reverse transcript can be introduced into to provide an interesting case of func- talk, Hughes discussed swapping experi- a naïve genome. tional mimicry. ments involving the plant homeodomain Tom Eickbush spoke about the evolu- PHD finger motif of the histone deme- tion, expression, and mechanism of reverse Targeting Specificity thylase JARID1A, the PHD finger and transcription of the R2 element, a site- In contrast to telomeres that are typically atypical bromodomain region of the his- specific TP retrotransposon that inserts confined to chromosome ends, the retro- tone methyltransferase MLL, and the into a conserved region of 28S ribosomal transcripts of viruses and transposons can chromodomain from polycomb group RNA of the insect Bombyx mori. R2 ele- target a plethora of sites. The host–element protein Cbx7. Fusions to the integrase- ments are present in most animal lineages interactions that govern the integration of binding domain of LEDGF were ex- and are transmitted vertically. They are reverse transcripts into host genomes by pressed in mouse cells that lack LEDGF. expressed as part of the 28S rRNA tran- a and a retrovirus-like trans- The results of these experiments demon- script. The 28S-R2 cotranscript is pro- poson are varied and fascinating. Dan strate that chromatin-binding domain cessed at the 5′ end by self-cleavage by Voytas spoke about integration specificity swapping in LEDGF redirects HIV-1 in- a double pseudoknot-bearing ribozyme in of the yeast retrovirus-like transposons, tegration to novel regions of the genome. the R2 RNA (30). The single R2 ORF Ty5 and Ty1. Ty5 preferentially targets The successful redirection of HIV-1 in- encodes a protein with RT, endonuclease, heterochromatic regions of the yeast ge- tegration events raises the possibility that and DNA binding domains. By following nome via an interaction between Ty5 in- lentiviral gene therapy vectors could be the TPRT activity of the purified B. mori tegrase and the silencing protein Sir4. As directed to nondeleterious regions of the R2 ORF, it was shown that the R2 ORF reported in the accompanying article (35), human genome. binds both the 5′ and 3′ end of R2 RNA, high-throughput DNA sequencing of a li- Regulation by Telomere Proteins and and it was proposed that RNA binding at brary of Ty5 integration sites in the Sac- Accessory Factors these sites coordinates first- and second- charomyces cerevisiae genome yielded fi strand cDNA synthesis (31). Eickbush a surprising nding: nearly one-quarter of The regulation of telomerase by auxiliary discussed his recent experiments identify- Ty5 retrotransposition events are euchro- components of the complex and telomere- ing RNA binding motifs in the R2 ORF matic. Further analysis of both hetero- bound proteins is an especially active area immediately N-terminal of the conserved chromatic and euchromatic insertions of investigation. Many such factors have RT motifs. Mutations in this region elimi- uncovered a secondary Ty5 target site, in been described in diverse systems. Kathy DNase I-sensitive, nucleosome-free re- Collins summarized her studies in eluci- nated TPRT activity, providing further – – support for a critical role for the RNA gions. In the accompanying article (35), dating the protein protein and protein template in the TPRT reaction. In- Voytas speculates that Sir4 brings the nucleic interactions within the Tetrahy- cDNA–integrase complex to the vicinity of mena telomerase RNP and between the terestingly, the position of the RNA bind- heterochromatin, but specific sites of in- RNP and telomeric DNA. Recent high- ing domains relative to the RT motifs in sertion are governed by the accessibility of lights include the identification and char- the R2 ORF are the same as the domains the DNA. DNA is most accessible in the acterization of a telomere adaptor of telomerase, named CP, QFP, and T, that nucleosome-free regions flanking ORFs; subcomplex and a replication protein A are necessary for RNP formation (32). hence both the primary and this newly (RPA)-related subunit (Teb1) that in- Since Eickbush’s discovery that the RT revealed secondary target site bias give teract with the core complex to form of the R2 TP retrotransposon uses the 3′- rise to Ty5 retrotransposition events that a holoenzyme with a high degree of repeat OH of nicked target DNA as its primer rarely disrupt essential coding sequences. addition processivity (38, 39). RPA is (4), scientists have noted the mechanistic This interpretation is bolstered by the a non–sequence-specific single-stranded similarities between retrotransposition and finding that ploidy has no effect on the DNA-binding protein complex that par- telomere addition. Indeed, a human L1 TP chromosomal distribution of Ty5, in- ticipates in numerous DNA transactions, retrotransposon lacking a functional dicating that coding sequences are not such as replication, repair, and recom- endonuclease (EN) domain can retro- necessarily cold spots because of selection bination. The RPA-like subunit in telo- transpose to dysfunctional telomeres (10). against deleterious events. In contrast to merase, however, exhibits a strong In his lecture and the accompanying arti- Ty5, Ty1 was shown to preferentially in- preference for single-stranded telomeric cle (33), John Moran described studies on tegrate into nucleosomal DNA upstream DNA and apparently exploits this high- the factors involved in initiating RT ac- of III-transcribed genes, a pol II gene- affinity binding to facilitate telomerase– tivity of the human L1 TP retrotrans- poor region. telomere interactions. In an accompanying fi – poson. Using partially puri ed RT RNA The human protein LEDGF, a bipartite article (40), Collins and collaborator Ming complexes from cells expressing L1 (34), protein consisting of integrase and chro- Lei (University of Michigan) report the Kulpa and Moran showed that L1 RT matin-binding domains, binds HIV-1 crystal structures of multiple oligonucleo- prebound to its RNA template uses DNA integrase and tethers the preintegration tide/oligosaccharide binding (OB) fold substrates that mimic telomeric ends as complex containing the HIV-1 reverse domains in Teb1 and examined their primers. Moreover, the L1 RNP, like te- transcript and integrase to genomic sites. roles in DNA binding and processivity en- lomerase, is associated with a nuclease The consequence of LEDGF tethering is hancement. The results offer a strikingly activity that can process the end of the that HIV-1 reverse transcripts typically detailed illustration of how the non– target DNA before reverse transcription is integrate within active transcription units sequence-specific RPA (presumably the initiated. The ability of EN-defective L1 (36). Steve Hughes and collaborators Alan ancestor of Teb1) may be adapted to serve RNPs to prime reverse transcription from Engelman (Dana-Farber Institute) and telomere-specific functions through the processed telomeric sequences in vitro and David Allis (Rockefeller University) have acquisition of altered DNA-binding the retrotransposition of EN-defective L1 fused the integrase-binding domain of properties. to telomeric ends in vivo imply that EN- LEDGF to chromatin-binding domains of The budding yeast S. cerevisiae, with its independent L1 RT activity resembles that other proteins (37) to test the idea that the constellation of genetic tools, has also of- of ancestral TP retrotransposons and HIV-1 target site bias is determined pri- fered a productive system for tackling te- telomerase. Thus, under defined circum- marily by the specificity of the chromatin- lomerase regulation. Three proteins of stances, the L1 RNP, like telomerase, binding domain of the host protein with importance are the telomere end-binding can carry out RNA-mediated DNA repair which HIV-1 integrase interacts. In his protein Cdc13 and the telomerase

Belfort et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20307 Downloaded by guest on September 27, 2021 regulatory proteins Est1 and Est3. All charomyces protein uncovered distinct transposition, was unexpected and re- three proteins are essential for telomere surfaces of the protein that mediate sep- markable (53). L1 retrotransposition in extension in vivo, but their precise mech- arate functions (47). In addition, Lundblad neural progenitor cells generates genetic anisms of action were debated (41). In described her study of an RPA-like tri- mosaicism that likely underlies the ex- a noteworthy development, an interaction meric complex named CST (Cdc13-Stn1- traordinary diversity of neurons. Gage between Cdc13 and Est1, which has long Ten1) that serves critical telomere-specific described how the down-regulation of the been postulated to serve the recruitment functions (51). The complex binds to the transcription factor Sox2 during the tran- of telomerase to telomeres (42), has at last terminal single-stranded, G-rich over- sition from neural progenitor cells to dif- been recapitulated in vitro. Ginger Zakian hangs and regulates numerous activities ferentiated neurons opens a brief window reported at the colloquium and in an that impact telomere length and structure for L1 mobilization to occur. Given the accompanying article (43) on a yeast (e.g., telomerase, recombination, and ability of L1 transposition to seed major overexpression system that enabled puri- nucleases). The repeated discovery changes in the genome, it is likely that fication of both Cdc13 and Est1, which of RPA-like proteins at telomeres sug- retrotransposition in neural stem cells is indeed interact specifically with each other gests a critical need to protect and/or highly regulated. Indeed, Gage and col- with a Kd of ≈250 nM. Intriguingly, mu- manipulate single-stranded DNAs at leagues have evidence for an unusually tations in CDC13 and EST1 that abolished chromosome ends. high level of L1 mobility in brain tissue of telomere maintenance in vivo did not have patients with two distinct neurological an obvious effect on this physical inter- Beyond the Genome: Retroelements disorders. The postmortem tissue from action, suggesting that a step other than During Development, Aging, and patients with Rett syndrome, a neuro- recruitment may be affected. Besides in- Disease developmental disorder arising from mu- teracting with Cdc13, Est1 has also been There is a growing awareness of retroele- tation of the MeCP2 gene, as well as implicated in promoting the assembly of ments as determinants of cell fate. The Ty1 induced pluripotent cells derived from Est3 into the telomerase complex (44, 45). LTR retrotransposon in S. cerevisiae is Rett syndrome fibroblasts, have approxi- Using a similar approach, Zakian and known to be a powerful player in genome mately twice the normal copy number of colleagues also demonstrated a direct and plasticity. Ty1 elements are associated L1 retrotransposons (54). In the accom- specific physical interaction between Est1 with DNA fragile sites, and reverse tran- panying article (55), Gage and colleagues and Est3, thus providing a plausible scripts of Ty1 and cellular mRNAs are extend this finding to brain tissue of pa- mechanism for how this assembly function frequently found at the junctions of ge- tients with ataxia telangiectasia (AT), is achieved (46). nome rearrangements. In her talk and the a disorder that results in mutations in In contrast to Est1, the action of Est3 is accompanying article (52), Joan Curcio the DNA damage-signaling gene, ATM. perhaps less controversial but more ob- reported that she and colleagues Patrick They show that the retrotransposition ef- scure. Intriguing similarities between this Maxwell (Rensselaer Polytechnic In- ficiency of a human L1 element endoge- protein and a domain of the mammalian stitute) and William Burhans (Roswell nously expressed in ATM-deficient human telomere binding protein TPP1 had been Park Cancer Institute) have found that cell lines or in ATM knockout transgenic noted earlier, although the functional and Ty1 retromobility increases during chro- mice is increased, suggesting that the evolutionary implications are unclear nological aging. This increase is associated higher L1 copy number in the brains of AT (47, 48). Although early experiments on with higher levels of chromosomal re- patients could be due to elevated retro- S. cerevisiae Est3 failed to disclose a contri- arrangements (detected via a loss-of-het- transposition. The findings indicate an as- bution of this protein to telomerase primer erozygosity assay) and chromosome loss sociation between overactive L1 retro- extension activity in vitro, both the Lund- in old cells. When retrotransposition is transposition and neurological disease, blad and Lue laboratories subsequently blocked by a variety of mutations or an RT although in both cases it remains to be reported stimulatory effects of Est3 from inhibitor, aging-associated genome in- determined whether unusually high retro- other budding yeasts, Saccharomyces cas- stability is diminished, and in some cases transposon copy number is the cause of tellii and Candida albicans (44, 49). At the lifespan is significantly extended. It is the neurological symptoms or a conse- colloquium and in an accompanying arti- possible that unrepaired retrotrans- quence of the underlying mutations. cle (50), Neal Lue presented studies of two position events lead to chromosomal Disruption of telomerase function is also unusual Est3 homologs in Candida para- rearrangements; alternatively, retro- responsible for a collection of human dis- psilosis and Lodderomyces elongisporus, transcripts synthesized by Ty1 may heal eases characterized by defective tissue which bear unique N- and C-terminal ex- spontaneous chromosome breaks, leading renewal, including aplastic anemia, pul- tensions not found in other Est3 homo- to DNA deletions, break-induced re- monary fibrosis, and liver cirrhosis. Per- logs. He reported robust interactions combination, or chromosomal rear- haps the prototype of such diseases is between these Est3s and the correspond- rangements. Although lifespan is a com- a heritable disorder known as dyskeratosis ing TEN domains, the putative “anchor plex phenotype influenced by a variety congenita (DC). DC patients suffer from site” of TERTs. Moreover, although Est3 of genetic and environmental factors, bone marrow failures and typically have alone does not bind DNA, it can be cross- Curcio’s work raises the possibility a characteristic triad of abnormal skin linked to DNA when associated with the that genome instability associated with pigmentation, nail dystrophy, and oral TEN domain. Thus, Est3 may potentially retrotransposition and RNA-mediated leukoplakia (56). The realization that DC extend the contact surface between the DNA repair mechanisms plays a role in is caused by telomere loss came initially telomerase holoenzyme and telomeric limiting lifespan. from the identification of disease-linked DNA. Although the precise mechanisms Human L1 elements, which constitute TERC (telomerase RNA) and DKC1 of Est3 and other regulatory factors re- ≈17% of the human genome, will, pre- (dyskerin, an RNA-binding telomerase main to be worked out, it now seems that dictably, be inherited by future gen- subunit) mutations in some patients (57). the regulation of telomerase is achieved by erations when they occur in germ cells. This conclusion was subsequently bol- domains and factors that modulate or However, the discovery by Rusty Gage and stered by the discovery of mutations in augment the RNP–DNA interactions. colleagues that L1 is mobile in human other telomerase subunit genes in distinct The mechanisms of Est3 were also neural progenitor cells in culture and that cohorts of patients. At the colloquium, discussed by Vicki Lundblad, whose ex- adult brain tissue, unlike other somatic Steve Artandi reported missense muta- tensive mutagenesis analysis of the Sac- tissues, sustains massive L1 retro- tions in yet another telomerase subunit

20308 | Belfort et al. Downloaded by guest on September 27, 2021 named TCAB1, which he had recently tumor stem cells) in a cell population (61). and functional relatedness of these retro- found to be required for the trafficking of Consistent with working through a non– elements contrasts with adaptations of the telomerase RNP to the Cajal body and telomere-related pathway, the effect was structure and mechanism that are tailored also for telomere maintenance (58, 59). not accompanied by telomere length al- to specific needs. Details of evolutionary It seems all but certain that continued terations and was abolished by knockdown relatedness of these diverse retroelements, studies of telomerase mechanisms and of BRG1 and TERT but not TERC (the contrasted with their distinctiveness, and assembly will have profound impacts on TER gene). It should be noted that the their role in determining cell fate are de- understanding the molecular basis and nontelomere functions of telomerase sub- scribed above and in the following articles. pathogenesis of telomere-related diseases. units have remained controversial and are In the journal Nature’s News and Views Beyond specific diseases, telomerase has seemingly at odds with the results of article written 4 decades ago, “Central also garnered attention as a major player in a number of transgenic mouse studies Dogma Reversed” (63), the prescient ed- cellular pathways that impact aging and (62). Nevertheless, the potentially dra- itor predicted that the discoveries of Bal- cancer. These connections have received matic implications of such functions in timore and Temin “are likely to generate compelling support from numerous cell- human diseases guarantee continued ex- one of the largest bandwagons molecular based and whole-animal studies (60). Al- perimentation and eventual resolution of biology has seen.” The editor went on to though many of the effects of telomerase these issues and arguments. ask, “Do uninfected eukaryotic cells or on cancer and aging, and hence on ge- bacteria contain similar RNA dependent nome stability, are clearly mediated Conclusions DNA ?” to those in virions. through its activities at telomeres, a num- A significant portion of most eukaryotic The meeting, celebrating the 40th anni- ber of intriguing studies have pointed to genomes is derived from RNA. Formation versary of the discovery of RT, fittingly “ ” extratelomeric functions of telomerase of this retrogenome involves the syn- keynoted by Dr. David Baltimore, an- subunits, especially the TERT protein. In thesis of reverse transcripts and the sub- swered this question. Similarly, this this vein, Bill Hahn explored the activity of sequent or accompanying incorporation of compendium of articles describes the an alternative complex formed by TERT, cDNA into the host genome. From the magnitude, complexity, and beauty of BRG1 (a chromatin remodeling ATPase foregoing it is clear that retroviruses, ret- the bandwagon. implicated in the transcriptional regula- rotransposons, and telomerase all played tion of many target genes), and NS/ a role in this genomic sculpting. Telo- ACKNOWLEDGMENTS. We thank Maryellen Carl GNL3L (two related proteins that are merase and TP retrotransposons can use and Rebecca McCarthy for their help with the highly expressed in proliferative, multipo- their RNA templates to synthesize novel manuscript, and Matt Stanger for rendering the figure. Work in the authors’ laboratories was tential cells). He found that over- DNA at the ends of chromosomal breaks supported by National Institutes of Health Grants expression of NS or GNL3L increased the and thus are capable of maintaining and GM39422 and GM4484 (to M.B.), GM52072 (to fraction of TICs (tumor initiating cells or molding the genome. The phylogenetic M.J.C.), and GM62631 (to N.F.L.).

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