Abstract
The amoeba Dictyostelium discoideum has a haploid genome in which two thirds of the DNA encodes proteins. Consequently, the space available for selfish mobile elements to expand without excess damage to the host genome is limited. The non-long terminal repeat retrotransposon TRE5-A maintains an active population in the D. discoideum genome and apparently adapted to this gene-dense environment by targeting positions ~47 bp upstream of tRNA genes that are devoid of protein-coding regions. Because only ~24% of tRNA genes are associated with a TRE5-A element in the reference genome, we evaluated whether TRE5-A retrotransposition is limited to this subset of tRNA genes. We determined that a tagged TRE5-A element (TRE5-Absr) integrated at 384 of 405 tRNA genes, suggesting that expansion of the current natural TRE5-A population is not limited by the availability of targets. We further observed that TRE5-Absr targets the ribosomal 5S gene on the multicopy extrachromosomal DNA element that carries the ribosomal RNA genes, indicating that TRE5-A integration may extend to the entire RNA polymerase III (Pol III) transcriptome. We determined that both natural TRE5-A and cloned TRE5-Absr retrotranspose to locations on the extrachromosomal rDNA element that contain tRNA gene-typical A/B box promoter motifs without displaying any other tRNA gene context. Based on previous data suggesting that TRE5-A targets tRNA genes by locating Pol III transcription complexes, we propose that A/B box loci reflect Pol III transcription complex assembly sites that possess a function in the biology of the extrachromosomal rDNA element.
Highlights
Mobile elements are obligate genomic parasites that amplify as selfish DNA and play important roles in driving the evolution of their hosts [1,2,3,4,5]
In this study we showed that retrotransposons tagged with the mbsrI gene can be followed to their natural integration sites, and that linear amplification PCR (LAM-PCR)-based transposon profiling is applicable to determine the integrome of mobile elements in the D. discoideum genome
The new data support our hypothesis that TRE5-A selects integration sites through interaction with the polymerase III (Pol III) transcription complex
Summary
Mobile elements are obligate genomic parasites that amplify as selfish DNA and play important roles in driving the evolution of their hosts [1,2,3,4,5]. Retrotransposons amplify by reverse transcription of RNA intermediates and integration of the resulting DNA copies at new locations of their host’s genomes. Non-LTR retrotransposons probably amplify by target site-primed reverse transcription (TPRT) [7, 8]. In this process, an element-encoded endonuclease cleaves one strand of the genomic DNA to generate a free 3’ hydroxyl that is used as a primer for reverse transcription of the retrotransposon’s RNA by the enzyme reverse transcriptase (RT) [9, 10]. Second-strand cleavage and cDNA joining to genomic DNA usually produce structural hallmarks of integrated retrotransposon copies, such as 5’ deletions, addition of non-templated nucleotides at 5’ insertion junctions, and variable-length target site duplications (TSDs) [11]
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