The regulation of gene expression at the RNA level is a major field of research in all organisms and this programme will provide training in an area currently rich in career opportunities. We are investigating two novel aspects of RNA control found in plants.
1) The role of the nucleolus in mRNA production and stability in Arabidopsis
We have generated evidence suggesting that the plant nucleolus is involved in nonsense-mediated decay (Kim et al. Plant Cell 21, 2045-2057, 2009). The eukaryotic nucleolus is responsible for the production of ribosomal RNAs and ribosome subunits. In addition, it is involved in a plethora of other RNA metabolism functions such as processing of tRNA, assembly of telomerase RNP etc. Unlike in animal cells, we found mRNAs in the plant nucleolus and analysis of these mRNAs in purified Arabidopsis nucleoli identified a high proportion of aberrantly spliced mRNAs which appeared to be substrates for nonsense-mediated decay (NMD). NMD is the process by which mRNAs containing premature stop codons (PTCs) by virtue of transcriptional or splicing errors or alternative splicing are recognised and degraded. We have also shown that some NMD factors are present in the nucleolus and have been investigating dynamics of proteins which interact with mRNAs (e.g. components of the exon junction complex – Koroleva et al. Plant Cell 21, 1592-1606). In addition mRNA transcripts with retained introns are not turned over by NMD. This raises the question of how specific transcripts are distinguished and how their subnuclear localisation affects their fate.
The project will involve the characterisation of the fate of various transcripts in the nucleolus of wild-type Arabidopsis and mutants in NMD and mRNA transport. The localization of different mRNAs and how this is affected by disruption of specific components of mRNA production and stability or the nucleolus itself will be examined to provide a model for the role of the nucleolus in mRNA production and NMD. Training will be given in basic molecular biology techniques (cloning, PCR, sequencing), RNA and expression techniques (RT-PCR and qRT-PCR); biochemical purification and fractionation of nucleoli and examination of their RNA and protein composition, sub-cellular and sub-nuclear localization. Regular written and oral presentation of research work will be an integral part of this studentship through lab meetings, departmental seminars, and at scientific meetings. Many opportunities for developing transferable, organizational and time-management skills are available.
2) The role of alternative splicing in regulating cold/drought stress responses in barley
Cold stress and drought adversely affects plant growth and development. Post-transcriptional regulatory mechanisms, such as pre-mRNA splicing, mRNA export and small RNA-directed mRNA degradation are emerging as key regulators of plant gene expression and play important roles in stress responses. Alternative splicing (AS) increases the proteomic and functional capacity of genes by producing alternative mRNAs derived from the same gene and ca. 42% of plant genes undergo alternative splicing. AS is also a key regulator of mRNA levels via the production of non-functional mRNAs which are degraded by NMD. AS is therefore a mechanism for tuning a plant’s response to environmental and developmental processes and we wish to examine the importance of AS in a crop species, namely, barley. Large scale EST analysis of the barley transcriptome has revealed ca. 500 alternative splicing forms and high throughput genome and transcriptome sequencing will rapidly generate AS information. AS is likely to be extensive and important in barley (e.g. key drought response transcription factors (DREBs) are alternatively spliced). The project will address the role of AS in cold/drought response pathways.