Telomerase is a ribonucleoprotein (RNP) enzyme that adds simple single-stranded DNA repeats onto linear chromosome ends to maintain chromosome stability and sustain cellular immortality. In humans, normal somatic cells lack telomerase activity and have limited replicative capacity, while near 90% of tumors reactivated telomerase to escape replicative senescence and attain cellular immortality. In adult stem cells, telomerase is expressed at an insufficient level, which leads to progressive telomere shortening and contributes to cellular senescence and tissue degeneration in the elderly. Moreover, mutations in telomerase have been linked to numerous telomere-mediated disorders such as dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary fibrosis. Understanding the inner workings of telomerase would provide keys to anti-cancer and anti-aging therapies.
Molecular Mechanism of Telomerase Action
One major focus of our research is to understand the molecular mechanism of telomerase function. Telomerase is a highly specialized reverse transcriptase that synthesizes telomeric DNA repeats at the ends of chromosome to confer cellular immortality. Unlike conventional RTs, telomerase RT contains an integral RNA component and uses only a very short region of the RNA as template. To add multiple DNA repeats processively, telomerase relies on a unique mechanism whereby the template RNA and the telomeric DNA dissociate and realign reiteratively during the telomere repeat synthesis. In spite of its essential role in tumorigenesis and aging, the detailed mechanism of telomerase action remains elusive.
Based on our recent studies, we propose a working model for template translocation. In our model, we hypothesize that, for the RNA template to translocate, the short RNA/DNA duplex must temporarily dissociate from the active site, undergo strand-separation and realign, leaving template available for next repeat synthesis. The RNA/DNA duplex must return to the active site for the next round of nucleotide addition. This research program aims to test the duplex-dissociation/binding hypothesis for template translocation using innovative assays that assess specific properties of the enzyme. These studies will provide important foundations for unraveling the mechanism of telomerase action.
RNA-Protein Interactions in Telomerase RNP
Telomerase functions as a ribonucleoprotein (RNP) enzyme requiring minimally the catalytic telomerase reverse transcriptase (TERT) protein subunit and the telomerase RNA (TR) subunit. Assembly of functional telomerase RNP relies on specific interactions between the TERT and TR components. Although a crystal structure of the TERT from the red flour beetle Tribolium castaneum has been determined, the Tribolium TR has not yet identified and very little is known about how the TERT protein interacts with the TR. We have recently established an in vitro assembly system for vertebrate telomerase RNP using T7 transcribed medaka TR and recombinant medaka TERT protein. The small size of the medaka TR and the mg scale of soluble medaka TERT purified make the medaka telomerase RNP an ideal system for X-ray crystallography study. With this useful system, we have recently mapped the RNA-protein binding interface in the conserved CR4/5 TR core domain and the TERT TR-binding domain (TRBD) by photoagent-dependent UV cross-linking and mass spectrometry to determine the cross-linking sites on the TERT surface. We are currently taking a similar approach to probe the TERT binding site for the TR pseudoknot domain which is highly conserved and also essential for telomerase activity.
Telomerase Biogenesis and Telomere Regulation in N. crassa and A. nidulans
Our recent identification of telomerase components from the non-yeast fungi offers a new opportunity to study telomerase function and telomere biology in the fungal model organisms Neurospora crassa and Aspergillus nidulans. Our data show that Neurospora and Aspergillus telomerases share a common TR core structure as well as many biochemical attributes with vertebrate telomerases such as processive addition of telomeric repeats and the use of vertebrate TTAGGG repeat sequence. In contrast, the commonly used budding and fission yeasts models appear to be more divergent in both telomerase structure-function and telomere biology. We have established the essential molecular genetic protocols in our lab for gene replacement and gene deletion in N. crassa and A. nidulans to study telomerase biogenesis and telomere regulation. We have also established powerful in vitro reconstitution systems for structure-function analysis of these fungal telomerase enzymes. The telomere length in N. crassa and A. nidulans is tightly regulated at 110-150 bp, significantly shorter than the 5-15 kb in human cells. In addition to understanding telomerase biogenesis, we will study telomere length regulation in N. crassa and A. nidulans. We are currently cloning telomere binding proteins and telomerase accessory subunits from N. crassa and A. nidulans to study how the telomere length is regulated in these non-yeast fungi.
Molecular Phylogeny of Telomerase RNP
The composition of telomerase RNP complex is remarkably divergent. In different species, telomerase RNA associates with different proteins and often shares distinct biogenesis pathways with other RNP enzymes. For example, in vertebrates but not ciliates or yeast, telomerase RNA associates with the dyskerin protein complex that is required for the biogenesis of both small nucleolar RNP (snoRNP) and telomerase RNP. The molecular mechanism that underlies the unusual course of telomerase RNP evolution remains a mystery. To better understand the diverse mechanisms of telomerase biogenesis and regulation, it is important to identify and characterize TR and its associated proteins from all major representatives of eukaryotes for phylogenetic studies.
The primary sequences of ciliate, vertebrate and yeast TRs are highly divergent and cannot even be aligned. Their secondary structures were thus determined independently by phylogenetic comparative analysis and show only limited similarity in the essential core domains. Due to the lack of sequence conservation, it has been extremely challenging to identify and clone TR genes from distantly related species using bioinformatics or conventional molecular biology approaches. We have recently developed a novel approach that combines biochemistry, RNA deep sequencing and bioinformatics to identify TR genes from a variety of eukaryotes. We have so far identified and cloned TR genes from teleost fish, filamentous fungi and recently sea urchin using this deep-sequencing based approach. We are currently cloning TR genes from a wide range of additional species to provide a framework for comparative studies of telomerase RNA structure as well as its functional roles in telomerase RNP assembly and regulation.