SILAC proteomics for quantitation of protein isoforms from alternative splicing in Arabidopsis seedlings

Genetic variation is an important basis for biodiversity and phenotypic variation. Plant growth and productivity and how plants respond to external stimuli such as pathogens/pests or stress conditions depend on the gene content of the plant species and the regulation of expression of the genes. Genes are regulated at many different levels. One important level is where genes are turned on or off or up or down - called transcriptional control. A second level occurs after the gene is transcribed or copied into RNA - called post-transcriptional control.

There are many different mechanisms of post-transcriptional control and alternative splicing (AS) is one of the most important. Alternative splicing is where different portions of a gene transcript are joined in different combinations to generate more than one messenger RNA (mRNA) from a gene. The resultant mRNAs can be translated into proteins with different functions or can be targeted for degradation. Thus, AS increases the proteome complexity of an organism and can regulate mRNA levels.

Alternative splicing affects many aspects of plant development, viability, adaptability to external conditions, metabolism and physiology and the most recent estimate suggests that at least 60% of intron-containing genes in plants undergo AS. More and more examples are being described where AS regulates expression or functional protein diversity. As our overall knowledge of this important regulatory level increases, it becomes necessary to be able to investigate the impact and dynamic changes of AS at the protein level. There are particular challenges in identifying and quantifying peptides deriving from different protein isoforms generated by AS (e.g. the abundance of peptides that distinguish specific isoforms from peptides common to isoforms)

Of various mass spectrometry (MS)-based quantitative proteomics methods, SILAC has a number of advantages for this specific application: many thousands of proteins are routinely detected, usually a significant fraction of proteins have multiple peptides, quantitation of proteins is relatively straightforward and good MS analysis software is available. In addition, SILAC,is able to quantify low abundance peptides and phosphopeptides, and analysis methods have been developed for analysis of protein isoforms in human cells.

SILAC has had very limited use in plants (cell culture) due to inefficient labelling with stable-isotope-containing amino acids as plants are autotrophic. We have developed a method to obtain high levels of SILAC labelling (>90%) and, more significantly, in Arabidopsis seedlings (as opposed to cell cultures). This makes this frontline technology amenable to whole plant systems for the very first time and means that it can be applied to a wide range of areas of plant biology from development to responses to biotic and abiotic stresses, and facilitates the comparison of mutants at the protein level.

In this proposal we will use this new modification of SILAC technology which allows it to be applied to plant seedlings to investigate alternative splicing at the protein level. We will analyse three sets of genetic lines where splicing factors or NMD factors are over-expressed or mutated and where we know from transcript analysis that the AS of many genes is significantly altered. These lines will maximise the opportunity for identifying isoform-specific peptides. There is currently very little data on protein isoform variants in plants and exploiting SILAC and using specific genetic lines will allow us to better understand the impact of AS at the protein level.

Finally, we expect our plant SILAC system to be applied more widely to other, non-model plant species and to provide a new method to investigate biochemical questions of post-translational modification, protein turnover, microRNA effects on protein levels, and protein interaction networks.

We have a great deal to learn about regulatory networks of AS in plants. One of the major challenges is to understand the impact and functions of alternative splicing at the protein level. The power of SILAC, its various biochemical applications and the recent development of methods to quantify protein isoforms in human cells make it an attractive quantitative proteomics system to study AS. SILAC has rarely been used in plants due to low efficiency of labelling. We now have routine, efficient SILAC labelling of Arabidopsis seedlings.

The goal of the project is to use SILAC to identify and quantify protein isoform-specific peptides. Two key aspects will maximise the identification of such peptides from the MS analysis. Firstly, we have extensive new data on novel splice junctions/transcript assemblies from an extensive RNA-seq analysis targeted at AS discovery in Arabidopsis. This data (and any new data) will be used to create a new peptide/protein database for MS searches. Secondly, we will use series of genetic lines where splicing regulators (RS31, GRP7) and NMD factors (UPF1) are over-expressed or mutated and where we know that AS of many genes is significantly altered (using our high resolution RT-PCR system). The NMD mutants are particularly targeted at identifying truncated proteins or peptides generated from AS transcripts (stabilised in the mutants) containing premature stop codons or upstream open reading frames. In addition, we will perform a limited RNA-seq on the SILAC samples to be able to relate peptide isoform data to AS transcript data and to specifically search for peptides on the basis of identifying genes where AS is significantly changed.

The College of Life Sciences is a centre where SILAC is being applied and analysis methods developed to address a breadth of biological questions. We have linked closely with the proteomics group and have access to newest technologies and expertise.

The research in this proposal is to deploy a frontline quantitative proteomics technology (SILAC) to plants (which has effectively not been possible before) and to address the important problem of impact of alternative splicing at the protein level. Although we have established SILAC in seedling for the first time with the view to investigate protein isoforms, this system will be much more widely utilisable by the plant community. We aim to advertise the fact that we have achieved routine SILAC labelling in seedlings by publishing our results in a timely fashion, by presenting this work at meetings. preparing short articles for publication in newsletters which have a wide constituency (e.g. the GARNet newsletter, European Plant Science Organisation (EPSO) newsletter) and putting the information of proteomic websites at the College of Life Sciences.

The research is clearly relatively far removed from direct or commercial application. However, the potential impact of the method of labelling seedlings is huge and we expect it to be taken up by many plant groups. More generally, the widespread nature of alternative splicing and the range of genes, pathways and processes involving AS (disease resistance, abiotic stress tolerance etc.) make it of growing interest not only to academic scientists but also to crop scientists and plant biotechnology companies working on phenotypic traits. AS is also likely to play a major role in allowing plants to adapt to their surroundings and will therefore also impact our understanding of genetic variation and ecological adaptation in plant/crop species.

The grant proposal is only for 12 months and therefore we will target dissemination of the outcomes of our research at national plant and RNA meetings and also in seminars given by the PI. To raise awareness of the importance of alternative splicing in plants, I have already organised a workshop at the 6th Symposium on Post-transcriptional regulation of Plant Gene Expression (Carry-le-Rout, France, 2007), a meeting session at the Society for Experimental Biology annual meeting (Prague, Czech Republic, 2010), and plant RNA workshops in Japan in July 2011 and Vienna in July 2012. We already have plans to hold a post-transcriptional gene regulation satellite meeting attached to the ASPB meeting in Providence, RI, USA at the end of July 2013.

We are also involved in educating undergraduates and postgraduates. In particular, the Plant Sciences division, with the James Hutton Institute, has established an MRes course on "Crops for the Future" where lectures on plant gene expression and RNA processing are put into a crop context. We have built a strong, international network of plant groups involved in gene expression and alternative splicing regulation and we will share our knowledge and host colleagues to transfer techniques.

We are active in public engagement through the University of Dundee, The James Hutton Institute and the Division of Plant Sciences who has built a relationship with the Dundee Botanical Gardens for plant exhibits focused at generating understanding of genetic principles.