Evolution of Poly-A Machinery in Plant Pathogens

BACKGROUND. The 3’ end formation of pre-mRNAs is a two-step process essential for eukaryotic gene expression1. First, pre-mRNAs are cleaved at their 3’ end. This step involves specific endonucleolytic cleavage at a canonical polyA site. The second step involves the polymerization of the adenosine tail by poly(A) polymerases. Several RNA elements contribute to polyA site recognition in animals.

It is common to find multiple potential 3’ end cleavage sites in eukaryotic genes, and the selection of the right polyA site is regulated in response to cellular cues2. This mechanism of alternative polyadenylation (APA) regulates the presence of cis elements in the mRNA and can generate different mRNA isoforms. Proteins involved in alternative polyadenylation include Cleavage Factor I in animals (CFIm) and Hrp1 in yeast3,4. Filamentous fungi (more complex organisms compared to unicellular fission and budding yeasts) have maintained both CFI and Hrp1 proteins suggesting that combined mechanisms regulate their polyadenylation process5.

OBJECTIVES. The overall objective of the proposed research study is to investigate the protein structure and components of the polyadenylation machinery in animals, plants, fungi and oomycetes. The specific objectives and expected targets are summarised below as follows. Using bioinformatics approaches, the student will carry out:

a) Identification of different protein complexes involved in 3’ end pre-mRNA processing in selected eukaryotic species with diverse life styles and environmental niches to gain insights into protein structure and evolution of the polyadenylation machinery in eukaryotes.
b) A survey for canonical and alternative polyadenylation signals using bioinformatic methods6,7 and available EST sequences from selected organisms. Transcriptome experiments will also be used to complete and confirm results.

Impact. Understanding mechanisms that regulate 3’UTR lengths by APA constitutes an uncovered area of research particularly in fungal pathogens of plants and animals, and generally in filamentous fungi. The cis elements present in the 3’UTRs such as microRNA target sites modulate gene expression by affecting cytoplasmic polyadenylation, subcellular localization, stability, translation and/or decay of the mRNA. Therefore, the selection of a proper 3´ end cleavage site represents an important step of regulation of gene expression. We expect to gain knowledge about the involvement of APA mechanisms in the expression of genes that help to adapt organisms to specific environmental conditions.

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2Wilusz, J. E. and Spector, D. L., An unexpected ending: noncanonical 3' end processing mechanisms. RNA 16 (2), 259 (2010).
3Guisbert, K. S. K., Li, H., and Guthrie, C., Alternative 3 ' pre-mRNA processing in Saccharomyces cerevisiae is modulated by Nab4/Hrp1 in vivo. PLoS Biol. 5 (1), 15 (2007). 
4Yang, Q., Coseno, M., Gilmartin, G. M., and Doublie, S., Crystal Structure of a Human Cleavage Factor CFI(m)25/CFI(m)68/RNA Complex Provides an Insight into Poly(A) Site Recognition and RNA Looping. Structure 19 (3), 368 (2011).
5Franceschetti, M. et al., Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3’ End Processing in Magnaporthe oryzae. PLoS Path. 7 (12), e1002441 (2011). 
6Tian, B., Hu, J., Zhang, H. B., and Lutz, C. S., A large-scale analysis of mRNA polyadenylation of human and mouse genes. Nucleic Acids Res. 33 (1), 201 (2005). 
7Doyle, F. et al., Bioinformatic tools for studying post-transcriptional gene regulation : The UAlbany TUTR collection and other informatic resources. Methods Mol. Biol. 419, 39 (2008).Phylogenetic analysis of the Poly-Adenylation machinery in plant pathogens