The aims of this grant are focused on a molecular dissection of the mechanisms by which yeast Tyl elements replicate their nucleic acids, how they integrate newly made Tyl cDNA into very specific genomic target regions, and how host functions participate in these processes. Replication of Tyl elements is mediated by host RNA polymerase II, the Tyl-encoded reverse transcriptase (RT) enzyme, and a cellular tRNA primer. Based on our extensive mutational analyses, we will probe the RNA structures that appear to be required for retrotransposition, and how they interact with the reverse transcriptase, the primer tRNA, and the packaging machinery. Integration of the resulting cDNA is mediated by the Tyl-encoded integrase. 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 Ill-transcribed genes. We seek to understand how this targeting is directed, presumably by a combination of Tyl-encoded and host functions. We will carry out genetic analyses of Tyl retrotransposon and host functions, supplemented by biochemical studies that exploit the in vitro systems we have previously developed for the study of Tyl reverse transcription and integration. Finally, we will explore the idea that elevating intracellular Mn+2 might interfere with HIV-1 replication. Based on the results of these experiments, we will pursue the isolation of compounds that raise intracellular Mn+2 in human cells by interfering with the human Pmrlp protein.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Genetics Study Section (GEN)
Program Officer
Rhoades, Marcus M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Johns Hopkins University
Schools of Medicine
United States
Zip Code
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