Retroviruses are important pathogens and useful therapeutic and biotechnology tools. Retrotransposons comprise almost half of the human genome with consequences for gene regulation and disease only beginning to be appreciated. Key lifecycle steps during which retroelement (retroviruses and retrotransposons) RNA, DNA and protein components interact with host macromolecular ribonucleoprotein complexes and nuclear pore complexes are poorly understood. Ty3 is a retroelement in budding yeast. It encodes Gag3 and Gag3-Pol3 proteins that assemble into virus-like particles (VLPs) and are subsequently processed into capsid, nucleocapsid, protease, reverse transcriptase, and integrase proteins with the activities of their retroviral counterparts. The RNA genome packaged into the VLP is reverse transcribed into cDNA which is translocated into the nucleus and integrated into host chromosomes. With parent GM33281 grant funding, our laboratory showed that Ty3 transposition is induced in mating cells. Translation of Ty3 RNA results in appearance of diffuse cytoplasmic Gag3 and Gag3-Pol3. Ty3 proteins and RNA collect within 2 h into cytoplasmic foci that contain components of ribonucleoprotein complexes known as RNA processing bodies (P bodies or PB). PB are cellular organelles that contain components of translation repressors, and RNA decapping, RNA degradation factors and microRNA processing, but the actual activities that occur within PB are poorly understood. We refer to these Ty3 assembly foci as Ty3-PB. We hypothesize that PB proteins provide important functions to transition Ty3 genomic RNA from translation template into packaged genome. Ty3-PB are physically associated with the nuclear pore complex (NPC) and this may facilitate entry of the Ty3 preintegration complex into the nucleus. The rapid induction of Ty3 RNA and dramatic clustering of translated Ty3 components and RNA with PB proteins into Ty3-PB followed by assembly of VLPs make Ty3 an ideal model for studies of PB function. We request Supplemental funds to use emerging technologies to study the dynamic interaction of Ty3 VLP and PB components. First, fluctuation correlation spectroscopy (FCS) with a National Research Resource Laboratory (UCI) will probe mechanisms of Ty3 RNA and protein and PB association into assembly complexes. Second, RNA-seq will be performed at the UCI Genomics High- Throughput Facility to map the association of ribosomes and Gag3 and PB proteins with Ty3 RNA. Third, a novel crosslinking reagent which can be reversed during mass spectrometry will be used in collaboration with L. Huang (UCI) to map interactions within and between cellular VLP, PB, and NPC. The Baldi group (UCI) has expertise in DNA-seq analysis and protein modeling and will collaborate on several aspects of this work.
Although retrotransposons comprise almost half of the human genome, the factors which influence retrotransposon intracellular assembly are poorly understood. This proposal uses the retrovirus like retrotransposon Ty3 in the model organism budding yeast in order to investigate the processes through which the retrotransposon RNA genome and structural proteins associate to form a virus like particle and the host proteins that contribute to that process. An exciting aspect of this proposal is the finding that assembly of thi retrotransposon is associated with a RNA granule which is implicated in control of retrotransposition and retrovirus restriction factors in animal cells.
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