This grant is focused on a molecular dissection of the mechanisms by which the yeast Ty1 elements replicate their nucleic acids, and how they integrate the newly made cDNA into very specific genomic target regions. Replication is mediated by host RNA polymerase II, the Ty1-encoded reverse transcriptase (RT) enzyme, and a cellular tRNA primer. Integration of the resulting cDNA is mediated by the Ty1-encoded integrase (IN). The process of integration is targeted to very specific regions of the host genome, namely """"""""integration windows"""""""" of several hundred base pairs immediately upstream of RNA polymerase III-transcribed genes. We seek to understand how this targeting is directed, presumably by a combination of Ty1-encoded and host functions. We will carry out genetic analyses of Ty1 retrotransposon and host functions, supplemented by biochemical studies that exploit the in vitro systems we have previously developed for the study of Ty1 reverse transcription and integration reactions.
The Specific aims are: 1) Molecular definition of the Ty1 reverse transcription pathway. We will comprehensively analyze the minimum cis-acting sites for Ty1 transposition, which are involved in packaging Ty1 RNA and priming its reverse transcription. We will analyze the mode of strong-stop DNA transfer, identify what plays the role of nucleocapsid protein in Ty1, and will continue efforts to develop a soluble in vitro system to study Ty1 reverse transcription. 2) Definition and characterization of the pathway leading to targeted integration of Ty1 upstream of pol III-transcribed genes. We will investigate the highly non-random nature of Ty1 integration, which occurs in specific """"""""integration windows"""""""". We will use a combination of genetic screens and biochemical studies using in vitro integration systems to identify important factors required for targeting. We will also biochemically analyze the Ty1 pre-integration complex and its localization to the nucleus. 3) Characterization of the host response to retrotransposition. We will begin new projects to explore the host transcriptional response to retrotransposition, and we will examine whether there is cell cycle regulation of transposition. We will examine the transcriptional interactions between Ty1 elements and the pol III genes that often lie adjacent to them.
|Taylor, Martin S; LaCava, John; Mita, Paolo et al. (2013) Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition. Cell 155:1034-48|
|Huang, Qing; Purzycka, Katarzyna J; Lusvarghi, Sabrina et al. (2013) Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription. RNA 19:320-32|
|Purzycka, Katarzyna J; Garfinkel, David J; Boeke, Jef D et al. (2013) Influence of RNA structural elements on Ty1 retrotransposition. Mob Genet Elements 3:e25060|
|Mularoni, Loris; Zhou, Yulian; Bowen, Tyson et al. (2012) Retrotransposon Ty1 integration targets specifically positioned asymmetric nucleosomal DNA segments in tRNA hotspots. Genome Res 22:693-703|
|Dai, Lixin; Taylor, Martin S; O'Donnell, Kathryn A et al. (2012) Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation. Mol Cell Biol 32:4323-36|
|Yarrington, Robert M; Richardson, Sarah M; Lisa Huang, Cheng Ran et al. (2012) Novel transcript truncating function of Rap1p revealed by synthetic codon-optimized Ty1 retrotransposon. Genetics 190:523-35|
|Dai, Lixin; Huang, Qing; Boeke, Jef D (2011) Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochem 12:18|
|Richardson, Sarah M; Nunley, Paul W; Yarrington, Robert M et al. (2010) GeneDesign 3.0 is an updated synthetic biology toolkit. Nucleic Acids Res 38:2603-6|
|Ivics, Zoltán; Li, Meng Amy; Mátés, Lajos et al. (2009) Transposon-mediated genome manipulation in vertebrates. Nat Methods 6:415-22|
|Scheifele, Lisa Z; Cost, Gregory J; Zupancic, Margaret L et al. (2009) Retrotransposon overdose and genome integrity. Proc Natl Acad Sci U S A 106:13927-32|
Showing the most recent 10 out of 62 publications