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An investigation into the role of post-transcriptional methylation of plant mRNA during development

Reference	BB/C513369/1
Principal Investigator / Supervisor	Professor Rupert Fray
Co-Investigators / Co-Supervisors	Dr Hongying Li
Institution	University of Nottingham
Department	Sch of Biosciences
Funding type	Research
Value (£)	253,402
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	30/09/2008
Duration	40 months

Abstract

Five approaches will be employed to investigate the role of plant mRNA methylation and of the mRNA methylase (AtMT-A70A): 1) Addressing the role of AtMT-A70A in plant development. We have shown that constitutive expression of AtMT-A70A cDNA can fully complement the embryo lethal phenotype. This cDNA will be fused to an embryo specific (J. Roberts personal com.) promoter and used to transform the hemizygous knockout line. If the construct driving embryo specific expression allows a homozygous insertion mutant to be obtained, any requirement for AtMT-A70A in developing roots or anthers (in which it is strongly expressed) should be revealed as an inability to form these organs or as a perturbation to their normal development. Concomitant with these experiments we will use root, tapetal and pollen specific promoters as well as an ethanol inducible promoter to drive tissue specific expression of a double stranded RNAi (all of these promoters are available to us). Again, we anticipate any developmental requirement for AtMT-A70A will be revealed as a perturbation of normal organ development. 2) We will identify the other proteins that associate with AtMT-A70A to confer the mRNA methylase holoenzyme function. MT-A70 requires additional, unidentified proteins for mRNA methylase activity in vitro. An Arabidopsis developing anther yeast two-hybrid Gateway library (Invitrogen ProQuest) as been constructed (Dr Zoe Wilson) and is available to us. We will use this to identify proteins that interact with AtMT-A70A. Interactions will be confirmed by epitope tagging both AtMT-A70A and the interacting proteins (Myc and HA tags) using binary gateway vectors. Following Arabidopsis transformation and crossing, we will test for co-immune precipitation. 3) Identification of mRNAs containing m6A. An antibody raised against m6A, will be linked to magnetic beads (Novagen) and used to enrich for m6A containing mRNAs from poly A+RNA extracted from auxin treated Arabidopsis roots and developinganthers. Probes will be generated from the m6A enriched and m6A depleted fractions and hybridised to the Arabidopsis ATH1-12501 GeneChip. Before proceeding with the chip hybridisations we will assess conditions for m6A enrichment by testing the ability to separate two in vitro transcribed RNAs from each other (one lacking and one containing m6A). For the first time, this method will allow the global analysis of m6A mRNAs and, in combination with conventional northern analysis, it will allow the m6A status of specific mRNAs to be monitored during growth and development. 4) Identification of m6A binding proteins. We will use a T7 phage display system (Novagen) to identify proteins that specifically interact with m6A containing RNAs. Pooled mRNA from auxin treated roots and developing anthers will be used to construct a phage display library. CDNA fusion to the capsid protein and controlling the ratio of native:fusion-capsid proteins allows the surface display of proteins in excess of 1000 amino acids. An RNaseI deficient E. coli strain is used as host when phage are selected for RNA binding. We will generate an m6A biotinylated transcript in vitro and link this to magnetic beads, an equivalent non-biotinylated transcript lacking m6A will be used as competitor. The library will be panned against the m6A RNA target in three rounds of enrichment to select m6A binding proteins. 5) Direct localisation of m6A within target mRNAs. We have shown that, under appropriate conditions, AMV reverse transcriptase preferentially stalls when opposite an m6A in the template. We will empirically test combinations of Mg2+, nucleotide concentrations and temperature as well as other RTs to develop this as a practical method for direct mapping of m6A within target mRNAs.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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