Intellectual Merit: The growth and development of eukaryotes is determined by complex interactions between their genetic make-up and the environment. In multicellular organisms, the different types of cells, tissues, and organs are ultimately defined by differential gene expression. Small RNAs (~20-30 nucleotides in length) play important roles in gene regulation as well as in defense responses against parasitic genomic elements such as transposons and viruses. The biogenesis and mode of action of these noncoding RNAs have attracted great attention in recent years, but many aspects of small RNA (sRNA) function are still poorly characterized. One central problem is our incomplete understanding of the factors involved in these processes. Interestingly, in the green alga Chlamydomonas reinhardtii, sRNAs perfectly complementary to a target transcript can inhibit protein synthesis without or with only minimal mRNA destabilization. This mechanism operates at a post-initiation step of translation and appears to alter the function/structural conformation of translating ribosomes so that they display reduced sensitivity to inhibition by some antibiotics such as cycloheximide. Evidence for sRNA-repressed transcripts remaining associated with polyribosomes, a strong argument in support of post-initiation translation inhibition, also exists in several other eukaryotes. The project will focus on the mechanistic characterization of this relatively unexplored process. It will involve the identification of molecular components of sRNA-guided effector complexes, involved in post-initiation translation repression, and the isolation and analysis of algal mutants defective in this process. The research will also characterize endogenous transcripts preferentially repressed by sRNAs at the translational level and begin addressing the role(s) of small RNAs in algal biology. Chlamydomonas offers an advantage for the isolation and characterization of novel factors involved in sRNA-mediated translation repression since the RNA interference (RNAi) machinery, despite being fairly complex, is not required for organismal viability. Moreover, novel mechanistic insights may be obtained through these studies because algae and land plants appear to lack orthologs of several of the proteins implicated in sRNA-mediated post-transcriptional regulation in metazoans. Characterization of RNAi in evolutionarily divergent eukaryotes is expected to contribute to our understanding of both common as well as lineage-specific mechanisms of sRNA-mediated silencing.
Broader Impacts The project will have an impact on human resource development through the direct training of undergraduate and graduate students and one postdoctoral fellow. Students will learn research methodology via hands on experience, with the goal of fostering their interest in scientific discovery. The laboratory findings will be used to stimulate the learning process and to promote discussions on the benefits of algal/plant science research in two plant biology courses. The training of a postdoctoral fellow for a career which combines research and education is also a central component of the project. Additionally, students will participate in a number of programs devoted to the instruction of minority individuals and in outreach activities with a non-profit organization working to educate the public on the nature and importance of science. The results of the project may also have practical implications for the genetic improvement of algal strains for biofuel production. One key constraint for the commercial development of algal feedstocks is the very limited knowledge on the regulatory mechanisms that control gene expression and metabolism. The anticipated findings will contribute useful information for understanding the function of small RNAs in microalgae and for the development of tools to modulate gene expression.
