Molecular mechanisms of integration site selection by Dictyostelium retrotransponsons
Final Report Abstract
In this project we aimed at investigating molecular mechanisms involved in the tRNA genetargeted transposition of tmobile elements from Dictyostelium discoideum, the long terminal repeat (LTR) retrotransposon DGLT-A and the non-LTR retrotransposon TRE5-A. In the experiments we investigated our hypothesis that integration preference at tRNA genes is a means to avoid gene mutagenesis in the compact D. discoideum genome. D. discoideum accommodates two related LTR retrotransposons: Skipper and DGLT-A. Using genome data of five Dictyostelid species, the recently published Physarum polycephalum genome, and a new genome sequence of a Protostelium species we established that Skipper and DGLT-A represent two related families of retrotransposons that are deeply rooted in amoebozoans and have distinct structural differences and integration preferences that may be influenced by the presence (Skipper elements) or absence (DGLT-A elements) of chromo domains in the carboxy-terminal parts of the integrase proteins. A hypothesis to be tested in future experiments is that the presence of a chromo domain tethers retrotransposons to heterochromatin, whereas the modification or loss of chromo domains may provide an opportunity to develop integration preferences in intergenic regions such as at tRNA genes. While investigating DGLT-A we obtained preliminary data explaining the integration preference upstream of tRNA genes by an interaction between the ribonuclease H domain (RNH) of DGLT-A with subunit TFC4 of the tRNA gene-specific transcription factor TFIIIC. This observation is interesting because position-specific integration of other LTR retrotransposons or retroviruses is usually mediated by the integrase protein. Using a genetically tagged DGLT-A we wanted to confirm the importance of the RNH-TFC4 interaction for integration site selection in vivo. Although this approach failed because we could not clone a retrotransposition-competent DGLT-A, we managed to generate instead a tagged Skipper element that may be useful in future experiments to study target site selection by chimeric Skipper/DGLT-A elements. In the process of developing tagged Skipper elements, we established that Skipper is completely silenced, in terms of retrotransposition activity, by the host cell. However, Skipper readily retrotransposes in mutant strains that lack the RNA-dependent RNA polymerase RrpC. Using genetically tagged TRE5-A retrotransposons we made progress in the genome-wide analysis of retrotransposon integration sites and we approached the question as to whether the targeting of tRNA genes is a means to avoid insertional mutagenesis of genes in the D. discoideum genome. While we observed that most of the ~400 tRNA genes in the D. discoideum genome can be targeted by TRE5-A, we also discovered a background integration activity of TRE5-A that is not targeted to tRNA genes but is instead scattered throughout the genome. This retrotransposition has attributes of endonuclease-independent integration, suggesting that TRE5-A may be involved in supporting the host to repair DNA strand breaks.
Publications
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(2014). The Dictyostelium discoideum RdRP RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the 3' spreading of small RNAs. Nucleic Acids Res. 42, 3330-3345
S. Wiegand, D. Meier, C. Seehafer, M. Malicki, P. Hofmann, A. Schmith, T. Winckler, B. Földesi, B. Boesler, W. Nellen, J. Reimegård, M. Käller, J. Hällmann, O. Emanuelsson, L. Avesson, F. Söderbom & C. Hammann
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(2015). The Physarum polycephalum genome reveals extensive use of prokaryotic two-component and metazoan-type tyrosine kinase signaling. Genome Biol. Evol., 8, 109-125
P. Schaap, I. Barrantes, P. Minx, N. Sasaki, R.W. Anderson, M. Bénard, K.K. Biggar, N.E. Buchler, R. Bundschuh, X. Chen, C. Fronick, L. Fulton, G. Golderer, N. Jahn, V.r Knoop, L. Landweber, C. Maric, D. Miller, A. Noegel, R. Peace, G. Pierron, T. Sasaki, M. Schallenberg- Rüdinger, M. Schleicher, R. Singh, T. Spaller, K.B. Storey, T. Suzuki, C. Tomlinson, J.J. Tyson, W.C. Warren, E.R. Werner, G. Werner-Felmayer, R.K. Wilson, T. Winckler, J.M. Gott, G. Glöckner & W. Marwan
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(2016). Convergent evolution of tRNA gene targeting preferences in compact genomes. Mob. DNA 7, 17
T. Spaller, E. Kling, G. Glöckner, F. Hillmann & T. Winckler
