Telomerase in Leishmania spp.: History
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Subjects: Biochemistry & Molecular Biology
Contributor:
Debora Silva
,
MARIA ISABEL N. CANO
,
Luiz Henrique Castro Assis
,
Marcelo da Silva

Leishmaniases are a  group of neglected tropical diseases caused by more than twenty different species of parasites of the genus Leishmania, presenting a variety of symptoms and degrees of severeness. These protozoans parasites are at the mainstream of biology/medical studies since understanding their biological particularities is crucial for the future development of antiparasitic therapies. Moreover, the comprehension of their cell cycle and its molecular mechanisms is of great value for the search of new possible treatments. In that sense,  telomeres emerge as a relevant subject in Leishmania spp. molecular biology research. They are the physical ends of eukaryotic chromosomes, granting stability to the genome and continued cell proliferation. Telomere elongation/maintenance is accomplished by a ribonucleoproteic complex called telomerase. The enzyme synthesis and adds repetitive telomeric sequences to the chromosome end termini using the 3’overhangs as substrates. In mammals, their gradual loss after many cell duplications can determine the cell fate by inducing replicative senescence or apoptosis. Here, we cover the main aspects of telomerase activity in Leishmania spp.

  • leishmaniases
  • telomeres
  • telomerase
  • Leishmania

1. Structure and Function

Telomerase is the ribonucleoprotein responsible for telomere elongation. The enzyme adds short repetitive sequences (e.g. 5’-TTAGGG-3’) at the single-stranded 3’G overhangs of each chromosome end[1]. Telomerase is essential to regulating telomere lengths and, consequently, for genomic stability. Telomerase comprises two main components, the protein Telomerase Reverse Transcriptase (TERT) and Telomerase RNA (TER), which are minimally required for the enzyme activity in vitro [2][3].

TERT is the catalytic component, and TER contains the template used by TERT to synthesize the telomeric repeats. In vivo, these two components form a complex with accessory proteins, which are necessary for biogenesis, enzyme activity, and nucleotide addition processivity [4][5][6]. The first report of telomerase activity in Leishmania spp. was reported by Cano et al. (1999)[7], and later, the enzyme purification and its biochemical characterization were described by Giardini et al., (2011)[8].

Telomerase activity was detected in all three parasite life forms (amastigote, procyclic promastigote, and metacyclic promastigote), presenting the canonical properties of other telomerases. However, the catalytic activity was shown to be temperature and life-stage dependent[9]. The gene encoding the Leishmania spp. TERT was characterized by Giardini et al., (2006)[10], and the TER component was described later by Vasconcelos et al., 2014[11].

In trypanosomatids, as in most eukaryotes, the TERT component is structurally composed of four structural/functional domains[4]: The Telomerase Essential N-terminal (TEN), Telomerase RNA-Binding Domain (TRBD), Reverse Transcriptase domain (RT), and the C-terminal extension (CTE). The TEN domain is involved in telomerase recruitment to the telomeres and enzyme processivity[12]. The TRBD interacts with TER and is connected to the TEN domain by an unstructured linker creating the RNA-binding pocket that binds single-stranded and paired RNA[13]. Both domains can also interact with proteins that stabilize the complex and help to recruit telomerase to telomeres and to regulate enzyme activity [14][15][16][17][18][19]. The RT is the catalytic core of the enzyme, which interacts with TER through the pseudoknot region[20], and is involved in the interactions with the RNA–DNA hybrid. Finally, the CTE domain stabilizes the RNA–DNA duplex, and differently from the other three domains, it is less conserved among different species[4]

Leishmania TERT preserves all the canonical domains found in other TERTs but shows some amino acid substitutions specific to the genus[10]. The knockout of Leishmania TERT seems to be harmful to the parasite since it induces a gradual decrease in cell density in the culture, apparent G1/G0 cell cycle arrest, morphological alterations, and telomere shortening (unpublished data).

The RNA component TER varies in length and sequence, presenting a conserved secondary structure in most eukaryotes. Variations of the TER size and sequence are observed among different organisms, and they are more prominent than TERT[21], which is conserved even among different taxa. In Leishmania spp., TER (LeishTER) is about ~2100 nucleotides long, and the mature molecule modified by trans-splicing contains a 5’ cap, a spliced leader sequence (SL), and a 3’ polyA tail. It also presents a conserved TBE (Template Boundary Element) motif C[U/C]GUCA in helix II, responsible for interacting with the TERT TRB domain and a C/D box snoRNA domain found in other TER[11]. LeishTER is expressed at similar levels in procyclic and metacyclic promastigote forms. The mature molecule coimmunoprecipitates and colocalizes with the TERT component in a cell cycle-dependent manner. Its secondary structure prediction shows the template sequence (5’->3’) in a single-stranded form localized near the 5’ end of the RNA molecule. Its double knockout (KO) led to partial cell cycle arrest and increased apoptosis in procyclic promastigotes. TER KO also triggers a progressive telomere shortening during continuous parasite passages (unpublished data). A similar effect was observed in T. brucei TER knockouts[22].

This entry is adapted from the peer-reviewed paper 10.3390/cells10113195

References

  1. Jerry W. Shay; Woodring Wright; Telomeres and telomerase: three decades of progress. Nature Reviews Genetics 2019, 20, 299-309, 10.1038/s41576-019-0099-1.
  2. Scott L. Weinrich; Ron Pruzan; Libin Ma; Michel Ouellette; Valeric M. Tesmer; Shawn E. Holt; Andrea G. Bodnar; Serge Lichtsteiner; Nam W. Kim; James B. Trager; et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nature Genetics 1997, 17, 498-502, 10.1038/ng1297-498.
  3. Tara L. Beattie; Wen Zhou; Murray Robinson; Lea Harrington; Reconstitution of human telomerase activity in vitro. Current Biology 1997, 8, 177-180, 10.1016/s0960-9822(98)70067-3.
  4. Abhishek Dey; Kausik Chakrabarti; Current Perspectives of Telomerase Structure and Function in Eukaryotes with Emerging Views on Telomerase in Human Parasites. International Journal of Molecular Sciences 2018, 19, 333, 10.3390/ijms19020333.
  5. Miriam Aparecida Giardini; Marcela Segatto; Marcelo Santos da Silva; Vinícius Santana Nunes; Maria Isabel Nogueira Cano; Telomere and Telomerase Biology. Progress in Molecular Biology and Translational Science 2013, 125, 1-40, 10.1016/b978-0-12-397898-1.00001-3.
  6. Lea Harrington; Biochemical aspects of telomerase function. Cancer Letters 2003, 194, 139-154, 10.1016/s0304-3835(02)00701-2.
  7. Maria Isabel Nogueira Cano; J. M. Dungan; N. Agabian; E. H. Blackburn; Telomerase in kinetoplastid parasitic protozoa. Proceedings of the National Academy of Sciences 1999, 96, 3616-3621, 10.1073/pnas.96.7.3616.
  8. M.A. Giardini; M.F. Fernández; C.B.B. Lira; M.I.N. Cano; Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage. Experimental Parasitology 2011, 127, 243-248, 10.1016/j.exppara.2010.08.001.
  9. Beatriz C. D. de Oliveira; Mark E. Shiburah; Stepany C. Paiva; Marina R. Vieira; Edna Gicela O. Morea; Marcelo Santos da Silva; Cristiane de Santis Alves; Marcela Segatto; Fernanda Gutierrez-Rodrigues; Júlio C. Borges; et al. Possible Involvement of Hsp90 in the Regulation of Telomere Length and Telomerase Activity During the Leishmania amazonensis Developmental Cycle and Population Proliferation. Frontiers in Cell and Developmental Biology 2021, 9, 1, 10.3389/fcell.2021.713415.
  10. Miriam A. Giardini; Cristina B. B. Lira; Fábio F. Conte; Luciana R. Camillo; Jair L. De Siqueira Neto; Carlos H. I. Ramos; Maria Isabel N. Cano; The putative telomerase reverse transcriptase component of Leishmania amazonensis: gene cloning and characterization. Parasitology Research 2006, 98, 447-454, 10.1007/s00436-005-0036-4.
  11. Elton J. R. Vasconcelos; Vinícius S. Nunes; Marcelo S. Da Silva; Marcela Segatto; Peter J. Myler; Maria Isabel Nogueira Cano; The Putative Leishmania Telomerase RNA (LeishTER) Undergoes Trans-Splicing and Contains a Conserved Template Sequence. PLoS ONE 2014, 9, e112061, 10.1371/journal.pone.0112061.
  12. Kathleen Collins; Single-stranded DNA repeat synthesis by telomerase. Current Opinion in Chemical Biology 2011, 15, 643-648, 10.1016/j.cbpa.2011.07.011.
  13. Susan Rouda; Emmanuel Skordalakes; Structure of the RNA-Binding Domain of Telomerase: Implications for RNA Recognition and Binding. Structure 2007, 15, 1403-1412, 10.1016/j.str.2007.09.007.
  14. Jinqiang Xia; Yun Peng; I. Saira Mian; Neal F. Lue; Identification of Functionally Important Domains in the N-Terminal Region of Telomerase Reverse Transcriptase. Molecular and Cellular Biology 2000, 20, 5196-5207, 10.1128/mcb.20.14.5196-5207.2000.
  15. Blaine N. Armbruster; Soma S. R. Banik; Chuanhai Guo; Allyson C. Smith; Christopher M. Counter; N-Terminal Domains of the Human Telomerase Catalytic Subunit Required for Enzyme Activity in Vivo. Molecular and Cellular Biology 2001, 21, 7775-7786, 10.1128/mcb.21.22.7775-7786.2001.
  16. Jing Huang; Andrew F Brown; Jian Wu; Jing Xue; Christopher Bley; Dustin P Rand; Lijie Wu; Rongguang Zhang; Julian J-L Chen; Ming Lei; et al. Structural basis for protein-RNA recognition in telomerase. Nature Structural & Molecular Biology 2014, 21, 507-512, 10.1038/nsmb.2819.
  17. Linnea I. Jansson; Ben M. Akiyama; Alexandra Ooms; Cheng Lu; Seth Rubin; Michael D. Stone; Structural basis of template-boundary definition in Tetrahymena telomerase. Nature Structural & Molecular Biology 2015, 22, 883-888, 10.1038/nsmb.3101.
  18. Jiansen Jiang; Henry Chan; Darian D. Cash; Edward J. Miracco; Rachel R. Ogorzalek Loo; Heather E. Upton; Duilio Cascio; Reid O’Brien Johnson; Kathleen Collins; Joseph A. Loo; et al. Structure of Tetrahymena telomerase reveals previously unknown subunits, functions, and interactions. Science 2015, 350, aab4070-aab4070, 10.1126/science.aab4070.
  19. Henry Chan; Yaqiang Wang; Juli Feigon; Progress in Human and Tetrahymena Telomerase Structure Determination. Annual Review of Biophysics 2017, 46, 199-225, 10.1146/annurev-biophys-062215-011140.
  20. Aaron R. Robart; Kathleen Collins; Human Telomerase Domain Interactions Capture DNA for TEN Domain-Dependent Processive Elongation. Molecular Cell 2011, 42, 308-318, 10.1016/j.molcel.2011.03.012.
  21. Emily D. Egan; Kathleen Collins; Biogenesis of telomerase ribonucleoproteins. RNA 2012, 18, 1747-1759, 10.1261/rna.034629.112.
  22. Ranjodh Sandhu; Samantha Sanford; Shrabani Basu; Mina Park; Unnati M Pandya; Bibo Li; Kausik Chakrabarti; A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei. Cell Research 2013, 23, 537-551, 10.1038/cr.2013.35.
  23. Luiz H. C. Assis; Débora Andrade-Silva; Mark E. Shiburah; Beatriz C. D. de Oliveira; Stephany C. Paiva; Bryan E. Abuchery; Yete G. Ferri; Veronica S. Fontes; Leilane S. de Oliveira; Marcelo S. da Silva; et al. Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?. Cells 2021, 10, 3195, 10.3390/cells10113195.
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