Investigators and Program Directors

Research Interests

Intracellular replication cycle of the
retrovirus-like transposable element,
Ty1, in budding yeast.

A chromosomal retrotransposon is
tagged in the 3' untranslated region
with an antisense retrotranscript
indicator gene, which contains an
antisense intron inside a marker
gene. Because the intron is spliced
only from the retrotransposon
transcript and not the marker
gene transcript, retrotransposition
events can be detected by a
simple change in phenotype.

Retroelements are mobile genetic elements that move around and between genomes through RNA intermediates. Retroelements are present in virtually all eukaryotes, where they influence genome content, organization and stability. Our laboratory is interested in the relationship between retroelements and the host genome in which they reside, or "retrogenomics".

Long terminal repeat (LTR)-retrotransposons and retroviruses evolved from a common ancestor. LTR-retrotransposons are transcribed by host proteins to produce retrotransposon mRNA, which is translated to produce structural (Gag) proteins and enzymatic (Pol) proteins. The retrotransposon proteins encapsulate the retrotransposon mRNA, and sometimes other mRNAs, to form virus-like particles (VLPs). VLPs contain reverse transcriptase, a polymerase that synthesizes DNA using mRNA as a template, and integrase, which inserts the new DNA copy into the host genome (Figure 1). We use the Ty1 LTR-retrotransposon in the yeast, Saccharomyces cerevisiae, as a model system for understanding the host-retroelement interface. We design and employ retrotranscript indicator genes (RIGs) to detect the formation of host sequences derived from RNA. RIGs are activated by loss of an intron during retrotransposition (Figure 2).

There are four active areas of research in our laboratory:

1. Activation of LTR-retrotransposons by DNA lesions. Over thirty different host proteins that repress Ty1 transposition (Rtt factors) have been identified in our lab, and many of these are highly conserved "genome caretaker" proteins, including telomerase. Work in our laboratory demonstrated that genome caretakers repress the formation of DNA lesions that trigger S-phase checkpoint pathways, which in turn induce the synthesis of Ty1 cDNA. For example, progressive shortening of telomeres in the absence of telomerase activates the DNA-damage checkpoint pathway, which in turn triggers Ty1 reverse transcriptase activity. We are using synthetic genetic array screens combined with secondary molecular assays to identify DNA-damage checkpoint effector proteins that interact with the Ty1 retrotransposition complex.
2. Formation of the retrogenome. Our lab is interested in the contribution of RNA to the content and organization of the eukaryotic genome. We are using an array of DNA replication associated mutants to understand how and when retrotransposons and other retrotranscripts are incorporated into the genome. RIGs have been inserted into the 3' untranslated region of different genes, allowing genetic selection for processed pseudogenes. Depending on their mechanism of incorporation into the genome, processed pseudogenes can replace protein-coding genes, resulting in intron loss and mutation, or they can give rise to gross chromosomal rearrangements. We are investigating the genetic factors involved in RNA-mediated chromosomal rearrangements.
3. Retrotransposition host factors. Our lab has used synthetic genetic array screens to identify a large collection of host proteins that are required for Ty1 retrotransposition. We are characterizing retrotransposition host factors (RHFs) that are required for VLP assembly and reverse transcription. Many of the RHFs that are necessary for cDNA synthesis are highly conserved proteins involved in mRNA localization, storage and degradation. Genetic, biochemical and microscopic approaches are being used to elucidate the role of conserved mRNA trafficking pathways are involved in VLP assembly and reverse transcription.
4. Human antiretroviral proteins. The human family of APOBEC3 cytidine deaminases includes potent inhibitors of retroviral replication. In collaboration with the lab of Bryan Cullen at Duke University Medical Center, our lab demonstrated that several human APOBEC3 proteins potently inhibit retrotransposition of Ty1 when expressed in yeast. The human APOBEC3G protein is incorporated into Ty1 VLPs via an RNA-dependent interaction with Gag, which is analogous to its incorporation into HIV-1 virions. APOBEC3G interferes with HIV-1 and Ty1 reverse transcription and edits HIV-1 and Ty1 cDNA. Given that Ty1 is only distantly related to the HIV-1 virus, our finding demonstrates that APOBEC3G inhibits a highly conserved step in the replication of LTR-retrotransposons and retroviruses. We are exploiting this system to identify and characterize host proteins that are required for APOBEC3 antiretroviral activity.

Contact Information

E-mail: joan.curcio@wadsworth.org
Phone: (518) 473-6078
Fax: (518) 474-3181