Epigenetic processes, which result in heritage changes in gene expression without modifications in DNA sequence, play important roles in the control of the development as well as in the cellular responses to viruses, and transposable elements. In fungi, plants, and animals the introduction of transgenes can lead to the silencing of homologous sequences by a variety of epigenetic phenomena operating at both transcriptional and post- transcriptional levels. The long-term goal of our research project is to understand the molecular basis of epigenetic gene silencing in eukaryotic organisms. Our immediate objectives are to identify and characterize genes involved in the post-transcriptional inactivation of transgenes and transposons in the unicellular green alga Chlamydomonas reinhardtii. Tagged and classical mutants defective in post-transcriptional silencing will be isolated by screening for reactivation of expression of a silenced transgene. The corresponding genes will be cloned by a variety of genetic and molecular approaches. The isolated mutants will also be tested for their effects on RNA interference, induced by transgenes designed to produce double-stranded RNA. Proteins interacting with a cloned DEAH- box RNA helicase, that is required for transgene and transposon silencing, will be identified by tandem affinity purification and by using the yeast- two hybrid system. The DEAH-box RNA helicase will be characterized in terms of cytological localization, enzymatic activities, and in vivo role(s) in different gene expression and in the cellular responses to RNA/DNA damage. Our findings will help to elucidate whether this helicase is involved in RNA processing and/or in a putative RNA surveillance system responsible for the degradation of aberrant RNAs. Using mutants defective in the transcriptional or post-transcriptional silencing of transgenes, the effect of these processes on the inactivation of transposable elements will also be evaluated. Transposon transcription will be examined by nuclear run-on assays ans transposon RNA stability will be determined following inhibition of transcription by Actinomycin D> These experiments will provide insights on the possible relationship between the mechanisms responsible for the transcriptional and post-transcriptional silencing. The overall findings are expected to contribute to our understanding of the molecular machinery involved in post-transcriptional gene silencing and RNA interference. If the silencing mechanisms are indeed effective as anti-transposon and anti-viral agents, their further elucidation will have impact not only in basic biology but also in medicine and agriculture.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062915-02
Application #
6520453
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
2001-05-01
Project End
2005-04-30
Budget Start
2002-05-01
Budget End
2003-04-30
Support Year
2
Fiscal Year
2002
Total Cost
$237,044
Indirect Cost
Name
University of Nebraska Lincoln
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
Kim, Eun-Jeong; Cerutti, Heriberto (2009) Targeted gene silencing by RNA interference in Chlamydomonas. Methods Cell Biol 93:99-110
Merchant, Sabeeha S; Prochnik, Simon E; Vallon, Olivier et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245-50
Cerutti, Heriberto; Casas-Mollano, J Armando (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50:81-99
Ibrahim, Fadia; Rohr, Jennifer; Jeong, Won-Joong et al. (2006) Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts. Science 314:1893
Sarkar, Nandita; Lemaire, Stephane; Wu-Scharf, Danxia et al. (2005) Functional specialization of Chlamydomonas reinhardtii cytosolic thioredoxin h1 in the response to alkylation-induced DNA damage. Eukaryot Cell 4:262-73
van Dijk, Karin; Marley, Katherine E; Jeong, Byeong-ryool et al. (2005) Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas. Plant Cell 17:2439-53
Rohr, Jennifer; Sarkar, Nandita; Balenger, Susan et al. (2004) Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J 40:611-21
Cerutti, Heriberto (2003) RNA interference: traveling in the cell and gaining functions? Trends Genet 19:39-46
Zhang, Chaomei; Wu-Scharf, Dancia; Jeong, Byeong-ryool et al. (2002) A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas. Plant J 31:25-36