PgmNr Y3072: Ty1 integrase interacts with RNA polymerase III-specific subunits to promote insertion of Ty1 elements into the Saccharomyces cerevisiae genome.

Authors:
S. T. Cheung 1,2 ; L. Ma 2 ; P. H. W. Chan 3 ; H. Hu 4 ; T. Mayor 1,3 ; H. Chen 4 ; V. Measday 1,2


Institutes
1) 1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada; 2) Wine Research Centre, University of British Columbia, Vancouver, BC, Canada; 3) Centre for High-Throughput Biology, University of British Columbia, Vancouver, BC, Canada; 4) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115.


Keyword: 02. Chromosome structure, variation, evolution and dynamics

Abstract:

The Ty1 retrotransposon belongs to a family of long terminal repeat elements in the Saccharomyces cerevisiae genome that replicates through an mRNA intermediate and is subsequently reverse transcribed into cDNA for genome insertion mediated by Ty1-encoded integrase (IN). Like many eukaryotic retroviruses, Ty1 insertions avoid the coding regions of the genome in order to prevent lethality of the host and propagate. Ty1 elements insert within a 1-2 kb region upstream of actively transcribed RNA Polymerase (Pol) III genes but the host factors that mediate Ty1 element insertion remained elusive for decades. We purified Ty1IN from yeast cell lysates using anti Ty1IN antibody-coupled magnetic beads followed by mass spectrometry analysis.  The Ty1IN co-purified proteins were enriched with peptides corresponding to multiple RNA Pol III subunits. To further investigate the interaction between Ty1IN and Pol III subunits, we purified Pol III using GFP-Trap Immunoprecipitation (IP). Co-IP experiments with multiple GFP-tagged Pol III subunits and Ty1IN showed that Ty1IN interacts with most Pol III-specific subunits, but not with shared Pol subunits.  However, since RNA Pol III is a stable complex and salt washes after GFP-Trap purification may not eliminate indirect protein interactions, we conducted an in vitro binding assay with purified Pol III-specific subunits and Ty1IN produced in E. coli.  We demonstrated that the Pol III complex purified from yeast and Rpc31, Rpc34 and Rpc53 purified from E. coli interact with Ty1IN in vitro. To further characterize the Pol III IN-binding domain we used a strain carrying an N-terminal truncation mutant of Rpc53, rpc53∆2-280 and found that Ty1 element insertion upstream of an integration hotpot, SUF16, a glycine tRNA gene, was ablated. Yet, the overall transposition frequency of the rpc53∆2-280 mutant strain was reduced by only ~25%, suggesting possible retargeting of Ty1 element insertions in the genome. Furthermore, we found that the interaction between Ty1IN and Rpc37, but not other Pol III subunits, is disrupted in the rpc53∆2-280 strain. This data suggests that Ty1IN may bind to multiple subunits of Pol III to mediate Ty1 integration independently. Together, we reported that Ty1IN interacts with RNA Pol III through the Rpc53/Rpc37 subcomplex, and plausibly via the Rpc82/Rpc34/Rpc31 subcomplex, to mediate insertion of Ty1 elements upstream of Pol III-transcribed genes. Since the structure of Ty1IN and the mechanism of genome integration are highly conserved among retrotransposons and eukaryotic retroviruses such as HIV-1, our study provides important insights into how INs seize host proteins for targeted genome insertion.