ASRNABIDOPHYS · Identification of light signaling components in the regulation of alternative splicing in plants
FP7 — People (Marie Curie Actions)
- Duration
- 2014-04-04 → 2016-04-03
- EU contribution
- €186,784
- Participants
- 1
- Scheme
- MC-IIF
Lines connect the coordinator with its partners.
Results in brief
Identification of light signaling components in the regulation of alternative splicing in plants
In order to survive, plants have to respond and adapt very quickly to various signals from the environment, in particular to changes in light quality and intensity. In fact, light is not only a source of energy for plants but also the main source of environmental imput that allows these organisms to finely adjust their gene expression pattern for maximizing their fitness. In plants, like in humans, the alternative splicing process is a major source for genetic diversity as a single gene might code for more than one protein, depending on which parts of the gene are kept to form a messenger RNA (the basis for the production of proteins). Alternative splicing has a significant impact on gene expression: It is estimated that over 90% of human genes and around 60% of Arabidopsis (model plant) genes are alternatively spliced. We recently showed that variations in light conditions affected alternative splicing of a subset of Arabidopsis genes via signals from chloroplasts. More specifically, nuclear alternative splicing events are responsive to changes in the photosynthetic electron transport chain that is in turn affected by light. We also showed that a signaling molecule travels through the plant as light regulation of alternative splicing is also observed in the roots, but only when the communication with the leaves (photosynthetic tissues) is not interrupted (see Petrillo et al., Science 2014 for further information). This project was conceived to identify the implicated genes and gene products in this light retrograde signaling pathway affecting the alternative splicing process in Arabidopsis thaliana. To achieve this goal we used 2 different strategies: on the one side, a long term unbiased mutant screening approach, which is defined by the generation of alternative splicing reporter constructs that would allow us to perform screenings for alternative splicing mutants and focus specifically in those mutants with defects in the light/dark regulation of splicing. On the other side, we were pursuing strategies to unravel the nature of the long distance signal that is originated in the chloroplast of the green tissue, and affects splicing of different nuclear transcripts in the roots. In this sense, we have now discovered the nature of the signal and also the sensor that acts in the root cells affecting RS31 splicing in response to light in the leaves.
Data: CORDIS, © European Union
Project objective
By analyzing the structure of eukaryotic genes it is possible to identify two different sequence types: those that are present in the mature transcript or mRNA, called exons, and those that are not present, called introns. RNA sequences which define exon/intron boundaries, spliceosome components, and splicing factors are represented among all eukaryotes. Besides these similarities there are some differences as intron/exon size, and the number of genes coding serine/arginine -rich (SR) proteins (splicing factors). These make plants ideal systems to study evolutionary conserved strategies for alternative splicing regulation, and to find novel mechanisms.During my PhD and postdoctoral research, we have shown that a retrograde signal generated in the chloroplast by light modulates alternative splicing. Interestingly, alternative splicing of the SR protein coding gene RS31 is severely affected. This project main goal is to identify the implicated genes, and gene products, in the light retrograde signaling pathway that affect RS31 alternative splicing. To achieve this aim I will generate a reporter construct with the coding sequence for the hygromycin phosphotransferase (Hpt), as selection marker, combined with the alternatively spliced region of RS31. Because of this reporter design, the synthesis of HPT protein would depend on the alternative splicing outcomes of RS31. Only those transgenic lines with higher amounts of alternative splicing isoforms which possess start codons (known as mRNA3 and mRNA2 for RS31) will be hygromycin-resistant. Since the abundance of these isoforms is very low in wild type plants exposed to light, these will be hygromycin sensitive in this condition, while mutants for RS31 alternative splicing would survive. By mutagenizing the transgenic lines genomes and by a further selection using hygromycin, I will be able to isolate alternative splicing regulatory factor mutants, and some of them will be involved in the retrograde signaling pathway.
Original text from CORDIS.
Participants
- MEDIZINISCHE UNIVERSITAET WIEN · WienCoordinatorAustria
Links
Data: CORDIS, © European Union