Telomerase elongates chromosome ends by addition of tandem telomeric repeats. This new DNA synthesis is required to balance the loss of DNA that is inherent in the incomplete replication of chromosome ends by conventional DNA polymerases. Single-celled eukaryotes constitutively activate telomerase and maintain a homeostasis of telomere length. Surprisingly, human somatic cells do not: they show progressive shortening of the telomeric repeat array with proliferation. Some human cells in the embryo, germline, epithelial tissues, and hematopoietic system have detectable levels of telomerase catalytic activity in cell lysates, but this level of activation is insufficient to prevent an overall loss of telomere length in all human tissues with age. Cumulative loss eventually produces a repeat array that is too short to protect the chromosome end, resulting in a forced exit from the cell cycle. Cancer cells dramatically up-regulate telomerase to permit indefinite growth. For this reason, telomerase inhibitors have great promise as broadly effective anti-cancer therapeutics. Telomerase activators may have equally significant application for expanding the renewal capacity of normal somatic cells with critically short telomeres arising from genetics, disease, age, or environment. The telomerase RNA subunit (TER) is expressed as a precursor that must be processed, folded, and assembled as a stable ribonucleoprotein (RNP) complex in order to accumulate to detectable level in vivo. This RNP then recruits telomerase reverse transcriptase (TERT) to generate the active enzyme. Collins lab efforts in previous funding periods have contributed pioneering insights about the endogenous pathway of human TER precursor processing and RNP assembly and discovered defects in the accumulation of mature telomerase RNP that underlie X-linked and autosomal dominant forms of the bone marrow failure syndrome dyskeratosis congenita.
The Specific Aims of the next funding period address remaining gaps in knowledge about human telomerase RNP accumulation and catalytic activation in vivo.
Aim 1 exploits methods of transient and stable TER expression in human cells to discover and characterize additional RNA motifs and proteins required for TER maturation and biological stability.
Aim 2 applies Collins lab expertise in RNA-protein interaction assays and affinity purification to define the biochemical defects that underlie inherited human diseases of telomerase deficiency.
Aim 3 investigates the assembly and activity of telomerase RNP with TERT. In vivo reconstitution methods will be combined with in vitro and in vivo activity assays to define TER motif functions in the catalytic cycle. The physiological specificity of RNA and protein domain interactions within the active RNP will be established. The long-term goal of these studies is to understand telomerase RNP assembly, catalytic activation, and cellular regulation in normal cells and disease and to exploit this understanding for improvement of human health.
Understanding the biochemical specificity of telomerase biogenesis and catalytic activation in human somatic cells and cancer cells will generate opportunities for clinical manipulation of telomerase to reduce cancer growth or enhance tissue renewal. In addition, therapies can be designed for patients with bone marrow failure syndromes arising from telomerase deficiency.
|Wu, Robert Alexander; Tam, Jane; Collins, Kathleen (2017) DNA-binding determinants and cellular thresholds for human telomerase repeat addition processivity. EMBO J 36:1908-1927|
|Chiba, Kunitoshi; Vogan, Jacob M; Wu, Robert A et al. (2017) Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo. Mol Cell Biol 37:|
|Wu, R Alex; Upton, Heather E; Vogan, Jacob M et al. (2017) Telomerase Mechanism of Telomere Synthesis. Annu Rev Biochem 86:439-460|
|Vogan, Jacob M; Zhang, Xiaozhu; Youmans, Daniel T et al. (2016) Minimized human telomerase maintains telomeres and resolves endogenous roles of H/ACA proteins, TCAB1, and Cajal bodies. Elife 5:|
|Wu, Robert Alexander; Dagdas, Yavuz S; Yilmaz, S Tunc et al. (2015) Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes. Elife 4:|
|Hockemeyer, Dirk; Collins, Kathleen (2015) Control of telomerase action at human telomeres. Nat Struct Mol Biol 22:848-52|
|Vogan, Jacob M; Collins, Kathleen (2015) Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle. J Biol Chem 290:21320-35|
|Katibah, George E; Qin, Yidan; Sidote, David J et al. (2014) Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5. Proc Natl Acad Sci U S A 111:12025-30|
|Wu, Robert Alexander; Collins, Kathleen (2014) Sequence specificity of human telomerase. Proc Natl Acad Sci U S A 111:11234-5|
|Sexton, Alec N; Regalado, Samuel G; Lai, Christine S et al. (2014) Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation. Genes Dev 28:1885-99|
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