We have long-term interests in understanding the ongoing evolutionary battle between genomic parasites and the host organism, and the cellular programs that have evolved in response to these conflicts. We study Long Interspersed Nuclear Elements (LINEs, or L1) due to their spectacular success in colonizing the human genome. L1s are retrotransposons, genetic elements that replicate through an RNA intermediate and integrate back into the chromosome. L1s are responsible for generating over one third of mammalian genome sequence, and L1 retrotransposition causes genome structural variation between human individuals. In addition, L1 is generally expressed in germ cells, and loss of L1 regulation is associated with sterility in mammals. This is likely due to the genotoxic effects of L1 retrotransposition. Since most cases of human sterility are not understood at the molecular level, this has potential significance for fertility research. We want to know how L1s replicate and how L1s are regulated. We are using a budding yeast (Saccharomyces cerevisiae) model to study unknown aspects of L1 retrotransposition. S. cerevisiae is well suited for this purpose;it is a preeminent organism for the study of genetics and cell biology of basic eukaryotic processes, and often the proving ground for the latest technologies in molecular biology. S. cerevisiae chromosomes are easily manipulated, providing great experimental flexibility, and the streamlined genome and proteome simplify the analysis of large data sets (relative to higher eukaryotes). The element we are specifically using in the budding yeast model is an L1 homolog from Candida albicans. We will clone and analyze retrotransposition insertions. We will explore the mechanism for circular retrotransposition product formation. We will investigate how L1 proteins recognize and bind to L1 RNA. We will examine the location and dynamics of L1 ribonucleoproteins (RNPs). We will also carry out genetic screens to identify host factors involved in the L1 replication cycle and ask whether meiosis represents a particular permissive "state" which is optimal for L1 RNP action. Finally, we will model the introduction and expansion of a family of L1 elements in a previously L1-naove host (budding yeast). Overall, these studies will provide insight into the mechanism of L1 replication and how L1s interact with a host cell.

Public Health Relevance

Our studies concern the replication and regulation of L1 elements, which are genetic elements that have produced over one third of mammalian genomes. These elements cause DNA damage and genome rearrangements, and when uncontrolled are associated with infertility in mammals. Our studies will help us understand the molecular details of how these L1 elements change our genome, how they interact with our cells, and how dangerous they are to our cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090192-03
Application #
8294975
Study Section
Genetic Variation and Evolution Study Section (GVE)
Program Officer
Janes, Daniel E
Project Start
2010-07-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$293,436
Indirect Cost
$105,336
Name
Carnegie Institution of Washington, D.C.
Department
Type
DUNS #
072641707
City
Washington
State
DC
Country
United States
Zip Code
20005
Annaluru, Narayana; Muller, Héloïse; Mitchell, Leslie A et al. (2014) Total synthesis of a functional designer eukaryotic chromosome. Science 344:55-8
Horn, Axel V; Klawitter, Sabine; Held, Ulrike et al. (2014) Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity. Nucleic Acids Res 42:396-416