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Structure, function and evolution of filamentous fungal telomerase RNA

Description

Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group,…

Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.

ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)

Created2011

The Adjuvant Properties of RNA Origami for Immunotherapy in a CT26 Cancer Model

Description

The properties of adjuvants to stimulate an immune response to treat cancer has sparked a major area of research in the field of immunotherapy. Given the presence of multiple RNA sensors in mammalian host cells for eliciting innate immunity, synthetic RNA nanostructures present a unique opportunity for adjuvant exploration. While…

The properties of adjuvants to stimulate an immune response to treat cancer has sparked a major area of research in the field of immunotherapy. Given the presence of multiple RNA sensors in mammalian host cells for eliciting innate immunity, synthetic RNA nanostructures present a unique opportunity for adjuvant exploration. While RNA nanostructures are organic and biocompatible in nature than other adjuvants, they could be tailored to have desired structural stability and functional diversity for in vivo application. In this study, a rectangular RNA origami nanostructure was designed to contain double-stranded RNA motifs and possess high structural stability. Using in vitro assays, RNA origami was shown to stimulate the toll-like receptor 3 (TLR3) signaling pathway, which has been reported to activate antigen presenting cells (APCs), natural killer (NK) cells, cluster of differentiation 8 (CD8) T-cells, and the secretion of proinflammatory cytokines. To explore RNA origami as an adjuvant for cancer immunotherapy, intraperitoneal administration of a murine colon cancer cell line (CT26) was used as a model system to mimic peritoneal metastasis (PM), in which RNA origami was investigated for its activities in mitigating PM tumor microenvironment and improving anti-tumor immunity. Given the poor outcome of the patients with PM and urgent need for new interventions, this study aims to translate the adjuvant activities of RNA origami demonstrated in vitro into potent anti-cancer immunotherapeutics. Here, it was shown that multiple intraperitoneal injections of RNA origami could inhibit tumor growth, leading to a significant delay and/or regression of metastatic tumor growth in the peritoneum. Furthermore, tumor-free mice, after being treated with RNA origami, were also resistant to a second challenge of tumor cells, indicating the development of the adaptive anti-tumor immunity. This immunity is dependent on T-cells since nude mice succumbed to tumor growth with or without RNA origami treatment. Thus, RNA-origami can function as an adjuvant to activate the innate immunity and subsequently the adaptive anti-tumor immunity, leading to tumor regression. Conceivably, RNA origami could be explored as an immunotherapeutic agent to improve the disease outcome of patients with peritoneal metastasis and peritoneal carcinogenesis.

ContributorsRodriguez del Villar, Ryan Luis (Author) / Chang, Yung (Thesis advisor) / Liu, Xiaowei (Committee member) / Qi, Xiaodong (Committee member) / Arizona State University (Publisher)

Created2018

A Self-Regulating Template in Human Telomerase

Description

Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR) component. It synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined, short template sequence from the integral TR. The specific mechanism of how the telomerase active site uses this short template region accurately…

Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR) component. It synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined, short template sequence from the integral TR. The specific mechanism of how the telomerase active site uses this short template region accurately and efficiently during processive DNA repeat synthesis has remained elusive. Here we report that the human TR template, in addition to specifying the DNA sequence, is embedded with a single-nucleotide signal to pause DNA synthesis. After the addition of a dT residue to the DNA primer, which is specified by the 49 rA residue in the template, telomerase extends the DNA primer with three additional nucleotides and then pauses DNA synthesis. This sequence-defined pause site coincides precisely with the helix paired region 1 (P1)-defined physical template boundary and precludes the incorporation of nontelomeric nucleotides from residues outside the template region. Furthermore, this sequence-defined pausing mechanism is a key determinant, in addition to the P1-defined template boundary, for generating the characteristic 6-nt ladder banding pattern of telomeric DNA products in vitro. In the absence of the pausing signal, telomerase stalls nucleotide addition at multiple sites along the template, generating DNA products with heterogeneous terminal repeat registers. Our findings demonstrate that this unique self-regulating mechanism of the human TR template is essential for high-fidelity synthesis of DNA repeats.

ContributorsBrown, Andrew (Author) / Podlevsky, Joshua (Author) / Qi, Xiaodong (Author) / Chen, Yinnan (Author) / Xie, Mingyi (Author) / Chen, Julian (Author) / Department of Chemistry and Biochemistry (Contributor)

Created2014-08-05

Prevalent and Distinct Spliceosomal 3 '-end Processing Mechanisms for Fungal Telomerase RNA

Description

Telomerase RNA (TER) is an essential component of the telomerase ribonucleoprotein complex. The mechanism for TER 3′-end processing is highly divergent among different organisms. Here we report a unique spliceosome-mediated TER 3′-end cleavage mechanism in Neurospora crassa that is distinct from that found specifically in the fission yeast Schizosaccharomyces pombe.…

Telomerase RNA (TER) is an essential component of the telomerase ribonucleoprotein complex. The mechanism for TER 3′-end processing is highly divergent among different organisms. Here we report a unique spliceosome-mediated TER 3′-end cleavage mechanism in Neurospora crassa that is distinct from that found specifically in the fission yeast Schizosaccharomyces pombe. While the S. pombe TER intron contains the canonical 5′-splice site GUAUGU, the N. crassa TER intron contains a non-canonical 5′-splice site AUAAGU that alone prevents the second step of splicing and promotes spliceosomal cleavage. The unique N. crassa TER 5′-splice site sequence is evolutionarily conserved in TERs from Pezizomycotina and early branching Taphrinomycotina species. This suggests that the widespread and basal N. crassa-type spliceosomal cleavage mechanism is more ancestral than the S. pombe-type. The discovery of a prevalent, yet distinct, spliceosomal cleavage mechanism throughout diverse fungal clades furthers our understanding of TER evolution and non-coding RNA processing.

ContributorsQi, Xiaodong (Author) / Rand, Dustin (Author) / Podlevsky, Joshua (Author) / Li, Yang (Author) / Mosig, Axel (Author) / Stadler, Peter F. (Author) / Chen, Julian (Author) / Department of Chemistry and Biochemistry (Contributor)

Created2015-01-01

Telomere Homeostasis Within Medaka (Oryzias Latipes)

Description

ABSTRACT Telomeres are vital in protecting chromosome ends to prevent telomere shortening. Telomerase is a ribonucleoprotein responsible for adding telomere repeats and maintaining telomere length. Telomerase holoenzyme consists of 2 major subcomponents: telomerase reverse transcriptase (TERT) and telomerase RNA (TR). The catalytic subunit is TERT and the subunit that adds…

ABSTRACT Telomeres are vital in protecting chromosome ends to prevent telomere shortening. Telomerase is a ribonucleoprotein responsible for adding telomere repeats and maintaining telomere length. Telomerase holoenzyme consists of 2 major subcomponents: telomerase reverse transcriptase (TERT) and telomerase RNA (TR). The catalytic subunit is TERT and the subunit that adds deoxyribonucleotide to the ends of chromosome is TR. TR contains an alignment portion and a template portion. Japanese Medaka (Oryzias latipes) has 4 nucleotide bases in its alignment region, which is similar to the 5-nucleotide bases in the human telomerase RNA alignment region. Because of the similar alignment region length, Japanese Medaka with 24 chromosomes was chosen to be used in this study. The question in this research was whether we could overcome heterogeneity. It was expected that when breeding short mean telomere length fish with another short mean telomere length fish, the new generation would have homogeneity. If short average telomere length fish and long average telomere length fish were to breed, the next generation fish would have heterogeneity in their average telomere length. In order to make a strong result statement further research needs to be done. The results from this study have somewhat supported the hypothesis, but will need additional information for a stronger validation. There were two inbreedings of short mean telomere length fish with another short telomere length; however, only one of the inbreeding pairs produced a fish with homogeneity (and supported the hypothesis). The other inbreeding pair depicted a large smear, a sign of heterogeneity. This may be due to a mutation in the subtelomeric portion. The method used to measure average telomere length was the terminal restriction fragment assay. Future research will involve using a different technique, quantitative fluorescence in sifu hybridizatrort to measure a more accurate telomere length of each chromosome.

ContributorsYee, Stephanie (Author) / Chen, Julian (Thesis director) / Stout, Valerie (Committee member) / Qi, Xiaodong (Committee member) / Barrett, The Honors College (Contributor)

Created2012-05