Telomere dynamics, genome stability and RNA processing
3 PhD projects offered in the IPP summer call 2019
Telomeres and Genome Stability
In most eukaryotic cells, telomeres are comprised of tandem arrays of short G-rich repeats bound by a conserved set of proteins. These nucleoprotein structures distinguish chromosome ends from DNA double-strand breaks and prevent DNA repair factors from inadvertently fusing the natural ends of chromosomes. Progressive telomere shortening and defects in telomere maintenance lead to genome instability, and are in part responsible for the gross chromosomal rearrangements that typify many cancers. Our overarching research goals are based on the firm belief that a better understanding of the dynamic interactions that occur at telomeres will ultimately enable us to identify compounds that modulate telomere length. Such reagents will have therapeutic use, either to limit the life span of tumour cells or to boost the proliferative potential of cell populations needed to maintain a healthy physiological homeostasis. The specific projects described here are guided by our fundamental interest in how chromosome ends are normally distinguished from DNA double-strand breaks.
PhD project 1: The molecular basis for chromosome end protection
Our lab has identified a region in the telomeric protein Rap1 from fission yeast that is important for end protection. Proteomics and genetics will now provide complementary approaches to elucidate the interactions that mediate the role of Rap1 in chromosome capping. Complementing the work in fission yeast, we have developed a protein-nucleic acid complex capture assay to analyze the composition of human DNA repair complexes at telomeric versus non-telomeric DNA ends. Intriguingly, despite the presence of hRAP1/TRF2 only on the former substrate, the NHEJ machinery readily assembles at both ends but is only active on one of them. In collaboration with Falk Butter and Petra Beli, the student will identify proteins that are specifically enriched at non-telomeric ends and assess their post-translational modification status. The student will then investigate whether their absence at telomeres is indicative of a critical distinction between protected and unprotected ends. By varying the in vitro assay conditions, we can examine the three double-strand break repair pathways (classical and alternative NHEJ and homologous recombination). This will allow the student to define the mechanisms by which each repair pathway is inhibited at telomeres.
PhD project 2: Environmental effects on chromosome capping
We have found that the requirements for chromosome capping in yeast are affected by the nutritional environment the cells are grown in. To gain further insights into condition-dependent sensitivity for telomere uncapping, the student will examine the extent to which DNA repair pathway activities vary in relations to growth conditions and nutrient status. The student will employ assays to monitor the repair of chromosome internal DSBs and telomere fusions, and combine telomere capture and mass spectrometry to assess whether chromosome end protection can be mediated by other factors than the canonical telomere binding machinery. Gaining a fundamental understanding of the requirements for end protection is of critical importance as even subtle increases in uncapping are significant in the context of triggering genome instability and cancerogenesis in multicellular organisms.
Pan L, Hildebrand K, Stutz C, Thomä N and Baumann P (2015) Minishelterins separate telomere length regulation and end protection in fission yeast. Genes & Dev 29: 1164–1174.
Bae N and Baumann P (2007) A RAP1/TRF2 complex inhibits nonhomologous end-joining at human telomeric DNA ends. Mol Cell 26: 323–334.
Sarthy J, Bae N, Scrafford J and Baumann P (2009) Human RAP1 inhibits hon-homologous end joining at telomeres. EMBO J 4: 3390–3399.
Telomerase, Aging and Cancer
Telomerase plays a critical role in determining the replicative lifespan of cells by replenishing terminal DNA sequences lost during normal replication. As telomerase is highly active in most cancer cells and is required for their continued proliferation, inhibition of telomerase has long been thought of as a promising avenue to treat a broad spectrum of cancers. Conversely, mutations in telomerase are associated with a group of degenerative diseases in which untimely depletion of stem cell pools limits tissue regeneration. These premature aging syndromes could be treated by increasing telomerase activity in stem cells. At its core, telomerase is comprised of a reverse transcriptase protein subunit and a long non-coding RNA. The latter provides the template for telomere repeat synthesis and forms the scaffold on which the protein subunits assemble. We are interested in deciphering how this large RNA is processed and folded to generate a functional scaffold. A thorough understanding of telomerase biogenesis and turn-over will ultimately allow us to identify compounds that modulate telomerase levels for therapeutic purposes.
PhD project 3: Telomerase biogenesis – one step at a time
Studies in our laboratory revealed a sequence of events that leads from transcription and polyadenylation of a telomerase RNA precursor to a processed RNA that is incorporated into the telomerase complex. This multistep process underlies extensive quality control with several decision points where further RNA assembly is in competition with degradation of the RNA. Our studies led us to the unexpected realization that the spliceosome has a previously overlooked function in 3’ end processing and identified several RNA binding proteins that have sequential roles in the assembly of functional telomerase. An incoming student will continue the characterization of the hierarchical assembly of telomerase that produces active enzyme by using a combination of biochemistry, Omics, bioinformatic analysis, molecular genetics and high-resolution microscopy.
Box, J.A., Bunch, J.T., Tang, W., and Baumann, P. (2008). Spliceosomal cleavage generates the 3' end of telomerase RNA. Nature 456, 910-914.
Tang, W., Kannan, R., Blanchette, M., and Baumann, P. (2012). Telomerase RNA biogenesis involves sequential binding by Sm and Lsm complexes. Nature 484, 260-264.
Kannan, R., Helston, R.M., Dannebaum, R.O., and Baumann, P. (2015). Diverse mechanisms for spliceosome-mediated 3' end processing of telomerase RNA. Nature Commun 6, 6104.
Paez-Moscoso, D.J., Pan, L., Sigauke, R.F., Schroeder, M.R., Tang, W., and Baumann, P. (2018). Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast. Nature Commun 9, 587.
Prof. Peter Baumann