Process-specific alternative splicing: a tool to monitor multiple alternative splicing events simultaneously in targeted plant genes

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

The majority of plant genes contain intervening sequences (introns). When a gene is turned on (transcribed), the DNA code is copied into a molecule of RNA called precursor messenger RNA (pre-mRNA). Intron sequences are removed from pre-mRNA by the process of splicing which joins the coding regions of genes (exons) together. The spliced mRNA is then translated into a protein. In many cases, in both plants and animals, pre-mRNAs can be spliced in different ways to generate different mRNAs / this is termed alternative splicing (AS). The alternative mRNAs produced can encode different proteins with different functions such that, for example, in humans, the 35,000 genes in the genome can give rise to more than 150,000 proteins. Thus, AS modulates gene function and expression and increases the number of proteins in higher eukaryotes. This flexibility allows the cells in an organism to fine-tune and subtly regulate cell activity. AS is not a random process but is highly regulated through the interaction of a large number of proteins called splicing factors with sequence signals in the pre-mRNA. Thus, in a particular cell type, the profile of splicing factors will determine the pattern of alternatively spliced transcripts of all of the genes being expressed. This will differ in different cell types, at different stages of development and in response to stimuli and, for example, stress conditions. To understand the regulation of gene expression at the level of alternative splicing, it is necessary to be able to measure changes in alternative splicing of multiple genes under different conditions. Over the last five years, estimates of the number of plant genes which undergo alternative splicing have risen from 7 to 35%. Despite at least a third of plant genes being alternatively spliced, little is known about how alternative splicing is regulated in plants. In particular, there is a need to better assess AS and its consequences, to address the co-ordinated regulation of AS in genes involved in the same biological process and to be able to examine cell- and tissue-specific alternative splicing. One of the major drawbacks currently is the lack of an accurate and reproducible system capable of monitoring multiple (10s to 100s) of AS events simultaneously. Research in animal systems has shown that alternative splicing is an essential aspect of gene expression with networks of alternative splicing regulation being superimposed on networks of transcriptional regulation. In plant systems, measuring global transcript levels is carried out routinely (transcriptomics) but alternative splicing and, in particular, the concept of co-ordinated and regulated alternative splicing has been largely ignored. We wish to establish a tool to monitor changes in alternative splicing of multiple plant genes in development and stress responses. The project will build collaborations between groups involved in aspects of developmental and stress biology of plants and the RNA biology/alternative splicing lab at the University of Dundee. The outcome of the project will be the demonstration that comprehensive process-specific AS RT-PCR panels can be used to accurately analyse changes in alternative splicing during development, under different conditions and in different mutant lines. By correlating patterns of changes in alternative splicing of specific genes or subsets of genes, information on the co-ordinated regulation of AS will be produced for the first time. Such information will be an integral part of systems approaches aimed at understanding interaction networks which regulate biological processes. The tool which we will develop will be of interest to plant scientists around the world. Although we will establish the tool by studying developmental processes and stress-induced genes in Arabidopsis, the system is very flexible and can be applied to examine any biological process in any plant species for which reasonable EST data exists.

Technical Summary

Alternative splicing increases the proteomic and functional capacity of genomes through the generation of alternative mRNA transcripts from the same gene. To understand gene expression in different cells under different conditions, it is necessary to address alternative splicing. Essential to this is 1) the systematic discovery of AS events in genes of interest, and 2) to have a system capable of measuring multiple alternative splicing events at the same time to identify changes in patterns of alternative splicing in different cells, tissues and organs, and in plants grown under different conditions. We will identify the key genes in specific plant processes with our collaborators and then identify all alternative splicing events both by bioinformatics and experimentally. The latter will require design of primers to cover the transcript, RT-PCR, cloning and sequencing of 50-80 cDNA clones. Specific primer pairs (with one fluorescently labelled primer) will then be designed to cover each AS event in all of the selected genes for a particular process. The AS RT-PCR panels will then be analysed using RNA isolated from plants or organs at different developmental stages, grown under different conditions or from specific mutants or plants over-expressing particular genes. RT-PCR products representing the alternatively spliced products are detected on an ABI3730 giving high resolution band separation, analysis of product size (+/-0.5nt) and intensity by GeneMapper. Significant changes in the ratio of the two alternatively spliced products detected by each primer pair will be determined by statistical analysis of three biological reps and control RNA from wild-type or untreated plants. Using RNA from micro-dissected or cell-sorted plant material, we will also begin to examine cell- and tissue-specific alternative splicing.

Publications

10 25 50
 
Description Key findings: 1) there is more extensive AS in plant genes than currently annotated; 2) the RT-PCR system here is sensitive and able to detect numerous AS transcripts even when they are expressed at low levels; 3) in future, expression analyses will have to take AS into account to obtain accurate measurments of expression.



Alternative splicing (AS) is where different messenger RNAs are produced from the precursor mRNAs of a gene. The AS isoforms either encode for a related but different protein which may have functional differences or are targeted for degradation thereby regulating expression of the gene. AS is highly regulated via the interaction of a large number of proteins called splicing factors with sequence signals in the pre-mRNA. Thus, in a particular cell type, the profile of splicing factors will determine the pattern of alternatively spliced transcripts of the genes being expressed. This will differ in different cell types, at different stages of development and in response to stimuli and, for example, stress conditions.



There must be cross-talk between transcriptional and AS regulation and recent results suggest that in plants there is only a small overlap between genes regulated at the transcriptional and AS level. Thus, many plant processes are likely to be regulated at both the transcriptional and AS levels. In addition, many plant AS events are as yet undiscovered. The objectives here were to identify AS events in genes in flowering time, cold response and root development pathways and combine this information with an accurate, high resolution method to measure changes in AS of these genes to provide information on "process-specific" regulation of AS.



We identified annotated AS events for genes in these pathways from Arabidopsis databases. Using a systematic analysis of the 134 genes selected with RT-PCR with multiple overlapping primers, cloning and sequencing, we confirmed many annotated events and found novel AS events. For the flowering time genes, 2-3 specific AS events are altered significantly between wild-type and fpa mutant plants suggesting a role for FPA in their splicing. Similarly, for the cold response genes analysed, annotated events were confirmed and novel AS events identified. The genes in the cold signalling pathway showed various AS events but the majority did not alter significantly with low temperature. We therefore demonstrated that many genes undergo AS events which are not annotated and therefore that a combined bioinformatic and experimental approach is needed. We have also identified that some of the AS events are regulated by changes in expression of SR splicing factors and that some AS transcripts are degraded by NMD.



The AS RT-PCR panels can be used to accurately analyse changes in alternative splicing during development, under different conditions and in different mutant lines. We will continue to expand the RT-PCR panel and have also had interest from other groups in our method for defining and measuring AS in sets of genes (e.g. core clock genes).
Exploitation Route The current application of the system is academic as we come to grips with how to integrate alternative splicing data with transcriptional data. The expertise in alternative splicing from this sytem is feeding into RNA-seq analysis programmes which will benefit plant and crop scientists.



In humans, a similar system (highly automated) is being used for diagnosis of specific conditions which are due to splicing defects (DNA level or trans-acting factor level) and ultimately diagnostic application in plants to detect abiotic/biotic stress could be The high resolution RT-PCR system is able to detect and accurately report on individual alternatively spliced isoforms. As such it is unique and of great utility in research aimed at answering questions about the regulation of expression at the level of alternative splicing.



To this end a number of research groups worldwide are currently collaborating to use the system. These include plant research groups from the UK, Austria, Poland, France, Germany, Israel, Portugal, Argentina and the USA. The projects cover the function of splicing factors and other proteins involved in mRNA biogenesis, mechanisms of alternative splicing regulation, regulation of and by the circadian clock, RNA-binding proteins in auxin response, genes involved in stress responses etc. The principles demonstrated by the 12 month grant have therefore led to substantial collaborations around the world which demonstrate the quality and innovative nature of UK science.



The high resolution system is also proving exceptional valuable in terms of validation of next generation transcriptome sequencing.



The system is also being applied to a crop species - barley.



A more general aspect of knowledge transfer has been to organise workshops on alternative splicing (attached to international meetings) to raise awareness of the importance of AS among plant scientists.
Sectors Agriculture

Food and Drink

Environment

 
Title High resolution RT-PCR for alternative splicing 
Description The method allows us to examine alternative splicing in plant genes; it gives qualitative (discovery of new events) and quantitative data; it has been established for Arabidopsis but we have translated this to barley; it is a system to validate RNA-seq analysis. 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact The method has been used by collaborators from around the world and has been established elsewhere by at least three other labs; it has led to many high quality pubications; 
 
Description Regulation of alternative splicing in plants 
Organisation Adam Mickiewicz University in Poznan
Country Poland 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Bielefeld University
Country Germany 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Leloir Institute
Country Argentina 
Sector Charity/Non Profit 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation Medical University of Vienna
Country Austria 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Institute of Plant Sciences (ISV)
Country France 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of Buenos Aires
Country Argentina 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of California, Davis
Country United States 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006
 
Description Regulation of alternative splicing in plants 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Andrea Barta (Vienna), Artur Jarmolowski (Poznan) and I were the only plant groups to be invited to join the European Alternative Splicing Network of Excellence. We established a joint project to develop a high resolution RT-PCR system to analyse alternative splicing in plant genes which had been established initially in my lab. This system was used by us and opened up to other scientists around the world to use in collaborative research. Andrea Barta and Maria Kalyna (currently at the Agricultural University of Vienna continue to collaborate actively.
Collaborator Contribution Different mutants and plant material grown under stress conditions were supplied for analysis; in many cases PhD students or postdocs came to Dundee to run samples on the high resolution Rt-PCR system
Impact Publications; Conference proceedings; organisation of international meetings
Start Year 2006