Source: UNIVERSITY OF ARIZONA submitted to NRP
COMPARATIVE EVOLUTION OF TELOMERASE FUNCTION IN PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1000115
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Plant Science
Non Technical Summary
In this proposal I outline experiments to identify and functionally characterize the telomerase RNA (TER), one of two minimal components of telomerase, from a group of plants closely related to the plant genetic model Arabidopsis. Data from these experiments will be used to determine if common structural elements and sequence motifs are retained in TERs from among this group of plants, and to refine bioinformatics approaches aimed at identifying TERs from across land plants. Moreover, plant TERs will be compared with other eukaryotic lineages to determine if structural homologies exist. Importantly, the work proposed here will help shed light on whether telomerase evolution is dramatically different in plants as compared with animals and fungi, and whether these differences are driven by a more rapid evolution of the TER sub-unit. Plants are much more tolerant of genomic instability than animals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011899104050%
2011899100050%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1899 - Oilseed and oil crops, general/other;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
Plants are much more tolerant of genomic instability than animals, perhaps made possible by the stability afforded by the telomerase activity, which in turn is controlled by the interactions between the telomerase RNA (TER) and reverse transcriptase component of telomerase (TERT). The proposed studies will shed light on whether telomerase evolution is dramatically different in plants as compared with animals and fungus and whether these differences are driven by a rapid evolution of TER. To address these questions it is necessary to (1) identify candidate TERs from additional plants, (2) test the ability of candidates to serve as a template for addition of the telomeric repeat by TERT, and (3) model secondary structures to reveal conserved elements that can serve as heuristics for the identification of TERs from other plant lineages.
Project Methods
Year One Objective 1: Identification of candidate TERs from additional plants. As indicated, Myc and HA tagged TERT plants have already been generated and immunoprecipitation approaches to identify SET 3 candidates can begin immediately. CLIP-seq will be performed during the first year. Objective 2: Functional tests for ability of candidates to serve as a template for addition of the telomeric repeat by TERT. Numerous candidates have already been generated using the first two strategies. These can be tested for function while candidate TERs are being isolated via immunoprecipitation experiments. In particular anti-sense TRAP experiments will begin. Objective 3: Secondary structure modeling to reveal conserved elements and development of a predictive tool for the identification of TERs more broadly in plants. Work associated with goal three of the proposal will be ongoing. Continual refinement of the bioinformatics algorithms will be in collaboration with the UGA RNA Informatics group. Year Two Objective 1: Identification of candidate TERs from additional plants. Analysis of CLIP-seq data is expected to be ongoing during year two. Objective 2: Functional tests for ability of candidates to serve as a template for addition of the telomeric repeat by TERT. Anti-sense TRAP experiments will be ongoing through the first two years as new candidates are identified. Any candidate TERs passing anti-sense TRAP screens can be immediately moved to expression analyses and mapping. It is expected that mapping may require some troubleshooting, but successful mapping of at least one candidate within the first two years of support is expected. Objective 3: Secondary structure modeling to reveal conserved elements and development of a predictive tool for the identification of TERs more broadly in plants. Work associated with goal three of the proposal will be ongoing. Continual refinement of the bioinformatics algorithms will be in collaboration with the UGA RNA Informatics group. Year Three Objective 1: Identification of candidate TERs from additional plants. Additional TER candidates are expected from refined bioinformatics approaches resulting from Objective 3. Objective 2: Functional tests for ability of candidates to serve as a template for addition of the telomeric repeat by TERT. Transgenic lines will be generated during the second summer and allow plants to be cultivated by students during year three. It should be noted that the identification of even a single candidate that passes both anti-sense TRAP and expression screens is likely to be a TER locus and its confirmation would increase by 100% the number of plants from which telomerase has been identified. Objective 3: Secondary structure modeling to reveal conserved elements and development of a predictive tool for the identification of TERs more broadly in plants. Work associated with goal three of the proposal will be ongoing. Continual refinement of the bioinformatics algorithms will be in collaboration with the UGA RNA Informatics group. Year Four Objective 1: Identification of candidate TERs from additional plants. Additional TER candidates are expected from refined bioinformatics approaches resulting from Objective 3. Objective 2: Functional tests for ability of candidates to serve as a template for addition of the telomeric repeat by TERT. Transgenic lines will be cultivated during year four. Cultivated lines grown in year three will be analyzed via PETRA. Phenotypes of late generation transformants will be documented. Objective 3: Secondary structure modeling to reveal conserved elements and development of a predictive tool for the identification of TERs more broadly in plants. Work associated with goal three of the proposal will be ongoing. Continual refinement of the bioinformatics algorithms will be in collaboration with the UGA RNA Informatics group. Year Five Objective 1: Identification of candidate TERs from additional plants. Completion expected in year 4. Objective 2: Functional tests for ability of candidates to serve as a template for addition of the telomeric repeat by TERT. Cultivated lines grown in year three will be analyzed via PETRA. Phenotypes of late generation transformants will be documented. Objective 3: Secondary structure modeling to reveal conserved elements and development of a predictive tool for the identification of TERs more broadly in plants. Work associated with goal three of the proposal will be ongoing. Continual refinement of the bioinformatics algorithms will be in collaboration with the UGA RNA Informatics group. Fine tuned predictive algorithm will be employed across land plants to generate putative TER loci.

Progress 10/01/13 to 09/30/18

Outputs
Target Audience:Scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project allowed graduate students and a post-doc attend scientific meetings to present their findings. In addition, the post-doc and one graduate student participated in career development opportunities through NSF. Recently the graduate student (Evan Forsythe) completed his PhD and moved to a post-doctoral position at Colorado State University. The post-doc (Andrew Nelson) supported on this project has now secured his own NSF funding and a new faculty position at Boyce Thompson Institute. How have the results been disseminated to communities of interest?Results have been disseminated via oral presentations, poster presentation at major scientific meetings, as well as in publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The sum or our biochemical and comparative molecular biological data led us to question the validity of the previously published telomerase RNAs. For some time we suspected that TER1 and TER2, identified by the Shippen lab at Texas A&M University (Cifeuntes-Rojas et al., 2011) were not correct. First, comparative analysis of genomic data from other Brassicaceae showed that the duplication yielding AtTER1 and AtTER2 occurred in A. thaliana, and thus all other species of Brassicaceae contained only a single locus orthologous to TER1 and TER2. In addition, we probed the biochemical activity and molecular evolution of components of telomerase across Brassicaceae and determined from the resulting data that the biochemical activity of different telomerase complexes as well as the component protein subunits are phylogenetically conserved, strongly indicating that the telomerase holoenzyme, including the telomerase RNA, should also be highly conserved in the family. Finally, we used CRISPR-Cas9 to delete the template domain at the TER1 locus of Arabidopsis. Importantly, these template mutants, which lack the critical domain required to synthesize the telomeric repeat at the chromosome terminus, showed no phenotype in assays that measure telomere length. These results contradict the findings of Cifuentes-Rojas et al. 2011, which first described the TER1 locus. All of these findings are documented in a manuscript prepared for submission to PNAS (see attached) and which definitively demonstrates that the true telomerase RNA remains to yet be discovered. In addition, we collaborated with the Shippen lab to probe the role of POT1, one of the putative components of the telomerase RNA and detailed these findings in a manuscript in Nucleic Acids Research (Arora et al., 2016). In addition to these findings, and due to our emerging view that AtTER1 is not the functional telomerase RNA subunit, we focused our energy in developing our bioinformatic pipeline for assessing the evolution of the telomerase RNA and other long non-coding RNA transcripts to aid in the identification of the true TER. These efforts yielded two publications. In Nelson et al., 2015 (G3) we use the pipeline to explore patterns of long non-coding RNA evolution across the family Brassicaceae, while in Nelson et al., 2016 (Frontiers in Genetics), we detail aspects of the workflow and provide downloadable executables as well as access through CyVerse. Evan Forsythe, a graduate student who was supported in part by this award, used the pipeline and developed phylogenetic expertise to collaborate with Dr. Danielle Reichert's group on the evolutionary history and functional diversification of cytochrome p450s in Arabidopsis (Liu et al., 2016, Nature Communications).

Publications


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:Scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Supported personnel attended a conference at Plant and Animal Genomes (January 2017). In addition, two personnel from the lab attended a career development workshop hosted by the National Science Foundation in Baltimore, Maryland. How have the results been disseminated to communities of interest?Publications and scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will complete RNA-seq experiments as outlined above. The project end date is approaching and these will be the final experiments under this project.

    Impacts
    What was accomplished under these goals? We are currently using an RNA-seq approach to identify TERs from Arabidopsis and Camelina. We have tested and optimized a two-part immunoprecipitation that relies on first obtaining highly purified nuclei using the INTACT system, followed by a second purification step via either tagged-Telomerase reverse transcriptase or a TERT antibody.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Evolinc: A Tool for the Identification and Evolutionary Comparison of Long Intergenic Non-coding RNAs. 2017. Nelson, A. D. L., Devisetty, U. K., Palos, K., Haug-Baltzell, A.K., Lyons, E and M. A. Beilstein. Frontiers in Genetics. doi.org/10.3389/fgene.2017.00052


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience: Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate students were trained in plant molecular biology and presented results from their experiments at the Undergraduate Biology Research Program annual meeting at the University of Arizona. Post-doctoral fellow involved in the project attended the Plant and Animal Genome conference and presented results of the bioinformatics pipeline developed with support under this project. How have the results been disseminated to communities of interest?Results have been disseminated through publications and presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Our goal is to use the immobilization technique we developed to identify other TERT interacting RNAs and thereby refine the model of telomere length maintenance in plants.

    Impacts
    What was accomplished under these goals? Over the past year we used CRISPR-Cas9 to alter the template domain of the previously described telomerase RNA (TER1) locus in Arabidopsis. We showed that deletion of this domain has no effect on the length of telomeres or telomerase activity. Our data indicate that TER1 cannot be the sole locus performing telomere elongation in Arabidopsis. In addition, we developed an immobilization technique to identify other RNAs that bind to the telomerase reverse transcriptase (TERT).

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: A genomic analysis of factors driving lincRNA diversification: lessons from plants. 2016. Nelson, A. D. L., Forsythe, E. S., Devisetty, U. K., Clausen, D. S., Haug-Batzell, A. K., Meldrum, A. M. R., Frank, M. R., Lyons, E., and M. A. Beilstein. G3. doi: 10.1534/g3.116.030338
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Evolution of Arabidopsis Protection of Telomeres 1 alters nucleic acid recognition and telomerase regulation. 2016. Arora, A., Beilstein, M.A., and D. E. Shippen. Nucleic Acids Research. doi: 10.1093/nar/gkw807
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A genomic and transcriptomic analysis of factors driving lincRNA diversification: lessons from plants. Non-coding RNA workshop, Plant and Animal Genome Conference, San Diego, CA, USA
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Convergent evolution of the telomerase RNA in Brassicaceae. Plant Genome Stability and Change, Tokyo, Japan


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience: The target audience includes other plant scientists working on telomere biology, as well as the broader scientific community working on telomere biology, genomic stability, genome structure, and evolution. Attendance at several different meetings communicated findings from supported research to members of these different communities. Changes/Problems:We have experienced some problems with the expression (translation) of our MYC tagged TERT lines. For this reason we have now also developed Flag tagged and GFP TERT lines. We are currently testing these in Arabidopsis. One other major change is the use of CRISPR-Cas9 to knock out expression of the TER1 locus in Arabidopsis. What opportunities for training and professional development has the project provided? Currently one post-doc and a graduate student are being trained as part of this project. Each mentors undergraduate students working in the lab, providing them an opportunity to improve communication skills. In addition, post-docs, grad students, and undergraduates participate in a weekly meeting with four other labs in the department and receive feedback about their experiments and progress. How have the results been disseminated to communities of interest?Yes, during the reporting period two manuscripts in high impact journals were published and a presentation of our most recent work was given at an international conference on plant genome evolution. What do you plan to do during the next reporting period to accomplish the goals? During the upcoming reporting period we will move forward using our newly developed INTACT lines for obtaining highly purified nuclei. Because telomerase is active in the nucleus, this should aid in our protein extraction of telomerase and subsequent identification of the TER in other species. Inaddition, we will identify a telomere mutant using our CRISPR-Cas9 plants that target the template region of TER1 in Arabidopsis. Finally, we will test additionalcontracts intext -/- null mutants to identify plants expressing epitope tagged versions of TERT for successful immunoprecipitation of TERT.

    Impacts
    What was accomplished under these goals? We have currently tested numerous TER candidates from Arabidopsis and have now developed Arabidopsis lines in which we can obtain highly purified nuclei. We are in the process of perfecting our immunoprecipitation protocols to pull down TERT in Arabidopsis and its closest relatives.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein-protein interaction. 2015. Beilstein, M. A., Renfrew, K. B., Song X., Shakirov E .V., Zanis M. J. and Shippen D.E. Molecular Biology and Evolution 32:1329-1341.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes of the Arabidopsis Nucleus. 2015. Gosai, S. J., Foley, S. W., Wang, D., Silverman, I. M., Selamoglu, N., Nelson, A. D.L., Beilstein, M. A., Daldal, F., Deal, Gregory, B. D. Molecular Cell. 57:376-388
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Evolution of Long non-coding RNAs: Lessons from Plants. Plant Genome Evolution / Elsevier Conference (Invited Oral Presentation). Amsterdam, Netherlands


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: The target audience includes other plant scientists working on telomere biology, as well as the broader scientific community working on telomere biology, genomic stability, genome structure, and evolution. Attendance at several different meetings communicated findings from supported research to members of these different communities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Currently one post-doc and a graduate student are being trained as part of this project. Both have attended meetings and presented their findings to the broader community during the funding period. Each mentors undergraduate students working in the lab, providing them an opportunity to improve communication skills. In addition, post-docs, grad students, and undergraduates participate in a weekly meeting with four other labs in the department and receive feedback about their experiments and progress. How have the results been disseminated to communities of interest? Yes, results have been presented at the international conference on Genetic Stability and Change in Plants held in Asilomar, CA, and at the annual meeting of the American Society of Plant Biologists. In addition, two major publications resulted from the funded work. What do you plan to do during the next reporting period to accomplish the goals? During the upcoming reporting period we will move forward characterizing the effect of knockdown of candidate TER loci in Arabidopsis. Furthermore, we will begin extraction of whole protein from floral tissue in our transformed lines from five different species of Brassicaceae. In the next year we will begin our RNA-seq experiments and begin the process of identifying additional TER loci. Identification of additional loci will permit us to address the final aim of the project which is to determine the secondary structure of TER in Brassicaceae and possibly more broadly in plants.

    Impacts
    What was accomplished under these goals? We have identifieda candidate TER from relatives of the plant genetic model Arabidopsis thaliana. Currently we are testing knockdown lines to determine if they show a reduction in telomere length, a hallmark of telomerase deficiency. Thus, the first two aims of the project are well underway. In addition, we are taking an RNA-seq approach to identifying RNAs that interact with the telomerase reverse transcriptase (TERT). To date we have transformed five species in the plant family Brassicaceae with TERT constructs containing an epitope tag (myc). We will immunoprecipitate TERT using an anti-myc antibody, extract RNA, and sequence. Using this approach along with a bioinformatic approach we developed we continue to screen for additional TER encoding loci among members of this important plant family.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Nelson, A. D. L., E. S. Forsythe, X. Gan, M. Tsiantis, and M. A. Beilstein. (2014) Extending the model of Arabidopsis telomere length and composition across Brassicaceae. Chromosome Research 22:153-166.
    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Beilstein, M. A., Renfrew, K. B., Song X., Shakirov E .V., Zanis M. J. and Shippen D.E. (In Press) Evolution of the telomere-associated protein POT1a in Arabidopsis thaliana is characterized by positive selection to reinforce protein-protein interaction. Molecular Biology and Evolution.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: The Continuing Evolution of the Telomerase RNA in the Plant Family Brassicaceae. Plant Genome Stability and Change (Contributed Poster), Asilomar, CA, USA.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Molecular Evolution of Brassicaceae Telomerase. American Society of Plant Biology Meeting 2014 (Contributed Poster), Portland, OR, USA.