Ambient temperature‐directed flowering time regulation : the role of alternative splicing
Verhage, Dina Sara Leonie - \ 2017
University. Promotor(en): Gerco Angenent, co-promotor(en): Richard Immink; Guusje Bonnema. - Wageningen : Wageningen University - ISBN 9789462579705 - 161
plants - flowering date - flowering - temperature - alternative splicing - molecular biology - genes - planten - bloeidatum - bloei - temperatuur - alternatieve splitsing - moleculaire biologie - genen
As a consequence of a sessile lifestyle, plants are constantly facing a fluctuating environment. In order to both profit maximally and protect themselves from these environmental cues, plants evolved ways to sense and respond to signals.
Ambient temperature is one of the cues for which plants have acquired a strategy to enhance their chance of survival and reproduction. Small changes in ambient temperature can have major effects on plant architecture and development, such as the transition from the vegetative to the reproductive flowering phase. The moment of flowering is an important event in the life cycle of a plant, since reproductive success depends on it.
In Chapter 1, I introduced the concept of alternative splicing, a molecular mechanism with a pivotal role in ambient temperature regulation of flowering time. In the model plant Arabidopsis thaliana, approximately 60% of the intron-containing genes show alternative splicing. Gene splicing varies depending on developmental stage and tissue type, but also environmental changes trigger differential splicing. Splicing is conducted by a large cellular machinery called the spliceosome, which recognizes intron-defining sequences and other cis-regulatory elements acting as splicing enhancers or silencers. Moreover, factors like chromatin structure, histone marks, RNA polymerase II (polII) elongation speed and the secondary structure of the pre-mRNA all play a role in the splicing outcome. Due to alternative splicing, a single gene can yield various transcripts. However, this does not cause an equal expansion of the proteome. Part of the transcripts are targeted for nonsense-mediated decay, or will be translated into unstable proteins. This is a way of regulating gene expression at the post-transcriptional or –translational level. Other transcripts will be translated into functional proteins that may be structurally and functionally different. Hence, alternative splicing creates additional complexity in the transcriptome, providing plants with molecular tools to respond to their environment, including the translation of ambient temperature alterations into a flowering time response.
Assessing the impact of alternative splicing on the diversity and evolution of the proteome in plants
Severing, E.I. - \ 2011
University. Promotor(en): W. Stiekema, co-promotor(en): Roeland van Ham. - [S.l.] : S.n. - ISBN 9789461730961 - 119
planten - genomica - rna - alternatieve splitsing - evolutie - plants - genomics - alternative splicing - evolution
Splicing is one of the key processing steps during the maturation of a gene’s primary transcript into the mRNA molecule used as a template for protein production. Splicing involves the removal of segments called introns and re-joining of the remaining segments called exons. It is by now well established that not always the same segments are removed from a gene’s primary transcript during the splicing process. The consequence of this splicing variation, termed Alternative Splicing (AS), is that multiple distinct mature mRNA molecules can be produced from a single gene.
One of the two biological roles that are ascribed to AS is that of a mechanism which enables an organism to produce multiple functionally distinct proteins from a single gene. Alternatively, AS can serve as a means for controlling gene expression at the post-transcriptional level. Although many clear examples have been reported for both roles, the extent to which AS increases the functional diversity of the proteome, regulates gene expression or simply reflects noise in splicing machinery is not well known.
Determining the full functional impact of AS by designing and performing wet-lab experiments for all AS events is unfeasible and bioinformatics approaches have therefore widely been used for studying the impact of AS at a genome-wide scale. In this thesis four bioinformatics studies are presented that were aimed at determining the extent to which AS is used in plants as a mechanism for producing multiple distinct functional proteins from a single gene. Each chapter uses a different method for analyzing specific properties of AS.
Under the premise that functional genetic features are more likely to be conserved than non-functional ones, AS events that are present in two or more species are more likely to be biologically relevant than those that are confined to a single species. In chapter 2 we analyzed the conservation of AS by performing a comparative analysis between three divergent plant species. The results of that study indicated that the vast majority of AS events does not persist over long periods of evolution. We concluded, based on this lack of conservation, that AS only has a limited impact on the functional diversity of the proteome in plants. Following this conclusion, it can hypothesized that the variation that AS induces at the transcriptome level is not likely to be manifested at the protein level. In chapter 3 we tested this hypothesis by analyzing two independent proteomics datasets. This type of data can be used to directly identify proteins present in a biological sample. Our results indicated that the variation induced by AS at the transcriptome level is also manifested at the protein level. We concluded that either many AS events have a confined species-specific (not conserved) function or simply produce protein variants that are stable enough to escape rapid turn-over.
Another method for determining whether AS increases the functional diversity of the proteome is by determining whether protein sequence variations that are typically induced by AS are common within the plant kingdom. We found (chapter 4) that this is not the case in plants and concluded that novel functions do not frequently arise through AS. We also found that most of the AS-induced variation is lost, similarly as for redundant gene copies, within a very short evolutionary time period.
One limitation of genome-wide analyses is that these capture only the more general patterns. However, the functional impact of AS can be very different in different genes or gene-families. In order fully assess the functional impact of AS, it is therefore important to also study the process within the functional context of individual genes or gene families. In chapter 5 we demonstrated this concept by performing a detailed analysis of AS within the MADS-box gene family. We were able to provide clues as to how AS might impact the protein-protein interaction capabilities of individual MADS proteins. Some of our predictions were supported by experimental evidence. We further showed how AS can serve as an evolutionary mechanism for experimenting with novel functions (novel interactions) without the explicit loss of existing functions.
The overall conclusion, based on the performed analyses is as follows: AS primarily is a consequence of noise in the splicing machinery and results in an increased diversity of the proteome. However, only a small fraction of the proteins resulting from AS will have beneficial functions and are subsequently selected for during evolution. The large remaining fraction is, similarly as for redundant gene-copies, lost within a very short evolutionary time period after its emergence.