Snapshot: Telomeres and Telomerase Andrea J

SnapShot: Telomeres and Telomerase Andrea J. Berman1,2 and Thomas R. Cech1 1HHMI and Department of Chemistry and Biochemistry, BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA; 2Present address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

Telomerase activity cycle: two types of processivity Protein component of telomerase is an RT 50 40 Active site TERT Approx. 30 size (kDa) Primer/telomere binding 5Ⱦ GGTTAG 20 18 HIV RT N C 70 CAAUCCCAAUC CAAUCCCAAUC 16 14 3Ⱦ 5 3 5 12 hTR Ⱦ Ⱦ Ⱦ 11 Fission yeast Trt-1 115 Disso Extension 10 c Nucleotide 9 iati 1 8 Dissociation on addition 7 Vertebrate TERT 130 processivity 6 5 5 2 Ciliate TERT 120 Ⱦ GGTTAGGGTTAG 5Ⱦ GGTTAGGGTTAG 4 CAAUCCCAAUC Translocation CAAUCCCAAUC 3 Repeat addition 3Ⱦ 5Ⱦ Yeast Est2p 100 processivity 3Ⱦ 5Ⱦ 2 NUMBER OF TELOMERIC REPEATS ADDED

1 TEN (N-terminal) domain RNA binding domain Reverse transcriptase domain 1

RNA components of telomerase and telomeric protection proteins vary among organisms

CILIATES VERTEBRATES CR4-CR5 domain O. nova H. sapiens/O. latipes/M. musculus TEBPβ 5Ⱦ 3Ⱦ p65 3Ⱦ 5Ⱦ TEBPα TIN2 TPP1

3Ⱦ 5Ⱦ 3Ⱦ Pseudoknot 5Ⱦ TRF1 TRF2 3 5 POT1 Template Ⱦ Ⱦ boundary T. thermophila Template Rap1

3Ⱦ AACCCCAAC 5Ⱦ H. sapiens/ Pseudoknot GGGTTG 3 O. latipes/ Template Ⱦ M. musculus Box H/ACA Template boundary domain PLANTS 5Ⱦ Template Pot1a Vertebrate TR 3’...... 5’ dyskerin, TCAB1 nop10, P8 gar1, nhp2 AUCCCAAAUC A. thaliana CCAAUCCCAAUC GGTTAG CR7 GGTTTAG Stn1/Ten1 domain

A. thaliana template region 5’ TRB/ 3’ TRFL/ 3’ TBP? 5’ CTC1 S. pombe template region

... 5Ⱦ 3Ⱦ... YEAST Template Template boundary Est1 arm ...Sm proteins ... Pseudoknot Terminal arm UCUCAUGCCAAUGUG GGTTACA GG S. cerevisiae pseudoknot- Template Template template domain boundary

S. pombe 3Ⱦ ACACACACCCACACCAC TGTGGGTGTG Stn1/Ten1 S. cerevisiae Poz1 Ccq1 5Ⱦ ... Ku arm Rap1 Tpz1 Rif1 Stn1/Ten1 Taz1 Rif2 5Ⱦ 3Ⱦ Cdc13 Assembly/stabilization/localization proteins 3 5 Pot1 5Ⱦ 3 Ⱦ Ⱦ Rap1 Ⱦ 3Ⱦ 5’ Points of contact between TERT and TR

1138 Cell 151, November 21, 2012 ©2012 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2012.11.008 See online version for legend and references. SnapShot: Telomeres and Telomerase Andrea J. Berman1,2 and Thomas R. Cech1 1HHMI and Department of Chemistry and Biochemistry, BioFrontiers Institute, University of Colorado, Boulder, CO 80309, USA 2Present address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

The very ends of eukaryotic chromosomes—the telomeres—have attracted a level of interest far exceeding the tiny fraction of the genome that they represent. Why this dispropor- tionate interest? First, telomeres are critical for genome stability and replication. Second, in most eukaryotes, telomeric DNA is replicated by a ribonucleoprotein (RNP) enzyme called telomerase. Although only a few RNP enzymes have been found in biology, e.g., the ribosome, RNase P, snoRNPs that modify rRNA, and snRNPs that contribute to mRNA splicing, they are fascinating biochemical entities that provide insights about RNA-protein interactions and clues about evolution from an RNA world. Finally, there is great medical interest in telomerase because it can confer cellular immortality. Inappropriate reactivation of telomerase in human cells contributes to oncogenesis, whereas even a 2-fold reduc- tion of telomerase is associated with the failure of proliferating tissues in dyskeratosis congenita and in cases of aplastic anemia and idiopathic pulmonary fibrosis.

Telomeres The most general functions of telomeres are to protect chromosome ends, preventing them from being treated as sites of DNA damage that are in need of repair, and to allow complete replication by providing a substrate for telomerase. At first glance, telomeric DNA seems disarmingly simple, consisting of multiple repeats of a short sequence encoded by the telomerase RNA. However, at the very ends of chromosomes, one of the two strands of the double helix continues as a 3′ single-stranded overhang, or “tail,” and this ssDNA can invade telomeric dsDNA to form a t-loop or it can fold on itself to form a very stable G quadruplex. The temporal orchestration of these structures and the mechanism of switching between them are important topics for future research. Many of the functions of telomeres are carried out by proteins that bind to the DNA sequence repeats. Some of these proteins are telomere specific, whereas others have roles throughout the genome. In Saccharomyces cerevisiae, the dsDNA-binding Rap1 protein has nontelomeric as well as telomeric functions and does not appear to interact directly with the ssDNA-binding Cdc13 protein. Mammalian telomeric DNA, in contrast, is capped off by the ssDNA-binding POT1-TPP1 heterodimer, which is bridged to the dsDNA-binding TRF1 and TRF2 proteins via TIN2. The entire complex, called shelterin, also includes the mammalian RAP1 homolog. The TPP1 protein is central to shelterin’s dual roles, simultaneously contributing to chromosome end capping and telomerase recruitment. The fission yeast S. pombe has provided an excellent system for understanding signaling at the telomere. Site-specific phosphorylation of the shelterin protein Ccq1 by the S. pombe homologs of the human ATM and ATR kinases promotes direct binding of telomerase. However, no Ccq1 homolog has been identified in mammals or budding yeast, so it is difficult to generalize this model.

Telomerase The “core enzyme,” the minimal unit required for catalytic activity, consists of the RNA subunit (which provides the template for DNA synthesis) and the catalytic protein subunit telomerase reverse transcriptase (TERT). In ciliated protozoa, an additional subunit (p43 in Euplotes, p65 in Tetrahymena) is essential in the core RNP because it functions as an internal chaperone to help fold the RNA and promote complex assembly. Telomerase holoenzymes contain additional proteins beyond the core enzyme, and these are involved in regulation and cellular trafficking. The TERT protein is evolutionarily related to other reverse transcriptases (RTs), such as retrotransposon and retroviral RTs, but is distinctly larger with two additional structural and functional domains. The RNA-binding domain (RBD) allows telomerase to maintain a stable complex with its RNA subunit; this distinguishes TERT from RTs that need to move along and copy an entire RNA element. The telomerase essential N-terminal (TEN) domain contributes to interactions with the telomeric DNA primer. The telomerase core enzyme reaction cycle is wonderfully intricate. The RNP binds the DNA primer (the chromosome tail) and the appropriate dNTP, catalyzes nucleotide addition, slides the primer-template by one base pair so that the TERT active site is available for the next reaction, sequentially adds nucleotides to complete one telomeric repeat (6 nt in mammals), and then translocates the template and repositions the primer to allow multiple repeat addition. Telomerase’s ability to synthesize multiple DNA repeats after a single primer-binding event is called repeat addition processivity. Telomerase RNA subunits vary in size from ~160 nt in Tetrahymena to ~450 nt in mammals to >1,000 nt in budding and fission yeast. The template region of the RNA has sequence complementarity to the telomeric DNA repeats; it binds the primer in an alignment region and then templates nucleotide addition, which can either be precise (ciliates, mammals, plants, and some budding yeast) or produce somewhat variable repeats (S. cerevisiae and S. pombe; in the latter case, the dotted line in the figure indicates two separate template alignment events that contribute to variable sequences in the repeats). Many telomerase RNAs have a template-adjacent stem loop that serves as a template boundary, providing a stop point for reverse transcription; though human telomerase RNA contains this boundary element, mouse telomerase RNA does not (dotted line in vertebrate RNA representation). The single-stranded RNA elements flanking the template help to position the template in the active site during translocation, and a pseudoknot/ triple-helix element contributes to catalysis. Peripheral RNA elements provide binding sites for accessory proteins that contribute to telomerase regulation or cellular localization. In S. cerevisiae, these are ever shorter telomeres 1 (Est1, the first telomerase component identified) required for telomerase recruitment/activity in S phase, the Ku heterodimer involved in nuclear localization and important for recruitment in G1, and the Sm ring involved in stability and cytoplasm-to-nucleus import. The RNA provides extended “arms” that bring these proteins into the complex; this RNA scaffold is “flexible” in the sense that the RNA arms do not have precise length, sequence, or positional requirements. The final holoenzyme component, Est3, binds to the yeast TERT (Est2) by protein-protein interactions; intriguingly, Est3 has structural similarity to mammalian TPP1. Telomerase holoenzyme components vary considerably through evolution. For example, the human enzyme has a 3′ RNA domain that resembles snoRNAs and binds two key sets of proteins, the dyskerin complex and TCAB1. The latter is essential for telomerase localization in Cajal bodies, a step that precedes telomerase action at telomeres. The Tetrahymena holoenzyme contains additional proteins that are required for high-processivity DNA synthesis.

Future Opportunities Although many groups have contributed to understanding telomerase assembly, function, localization, and structure since the discovery of telomerase by Greider and Blackburn in 1985, there is still much to be learned. Remaining questions include those of telomerase regulation, the function of specific regions of the RNA in the catalytic cycle, and determining the structure of a telomerase holoenzyme. The contribution of telomerase activation to oncogenesis is incompletely understood, and the extent to which telomerase insufficiency may contribute broadly to stem cell failure and aging deserves further exploration. Certainly the next decade of telomerase research will lead to exciting discoveries and will shed more light on this critically important RNP.

References

Du, H.-Y., Bessler, M., and Mason, P.J. (2008). Telomerase mutations and premature ageing in humans. In Telomeres and telomerase in ageing, disease and cancer, K.L. Rudolph, ed. (Berlin: Springer-Verlag).

Egan, E.D., and Collins, K. (2012). Biogenesis of telomerase ribonucleoproteins. RNA 18, 1747–1759.

Podlevsky, J.D., Bley, C.J., Omana, R.V., Qi, X., and Chen, J.J. (2008). The telomerase database. Nucleic Acids Res. 36 (Database issue), D339–D343.