Regulation of gene activity is of fundamental importance for the ability of cells to control growth, differentiation and adaptation to environmental change. Genes contain coded information on production of biologically active macromolecules whose synthesis is the result of a series of linked molecular processes. These processes begin with transcription of the gene, i.e. production of an RNA copy of the DNA, and control of gene transcription is, therefore, one of the most important points of control of gene activity altogether. Results from studies of multiple organisms, in particular mammalian cells, have shown that genes of higher organisms are flexible units that can give not only to different quantities of RNA copies, but also to qualitatively different RNA copies, sometimes with important ramifications for the final gene activity. The choice of the location of transcription start sites constitutes an important element of this flexibility of genes.
Precise studies of transcription start sites in mammalian cells have also established an interesting and fundamental property of the process of transcription itself: In these cells, transcription occurs in both directions around genes, but only transcription into the gene, not the divergent non-coding transcription, gives rise to stable RNA, as the divergent non-coding transcripts are rapidly degraded by an RNA degradation complex called the RNA exosome.
In plants, only few studies have attempted to identify transcription start sites genome wide, and it is, for instance, still unclear whether transcription in plants is generally bidirectional or not. The objective of this thesis is to (i) produce solid data on transcription start sites in the model plant Arabidopsis thaliana, either wild type or mutants with lesions in genes encoding RNA exosome factors, (ii) analyze these data to clarify whether bidirectional transcription occurs, and (iii) clarify whether alternative choice of transcription start sites may be of functional importance in rapid genetic reprogramming, studied here in connection with activation of the innate immune response.
My work shows that contrary to mammals, bidirectional transcription is rare, albeit not completely absent in Arabidopsis. I also show that a likely explanation for this fundamental difference may be that plants have a more versatile machinery for production of small regulatory RNA than mammals: In Arabidopsis, divergent transcripts are readily converted into small regulatory RNAs, potentially leading to aberrant repression of gene activity at the transcriptional level. My work also shows that antisense transcription initiating in downstream parts of genes is relatively widespread in Arabidopsis, and that the RNA resulting from such transcription initiation events is normally degraded by the exosome. Interestingly, this type of antisense transcription tends to occur in genes encoding regulatory factors, suggesting functional importance. These results have been summarized in a paper that has been positively evaluated at the top journal The Plant Cell.
The second part of my work shows that changes in transcription start sites occur quite frequently in genes induced during the activation of the immune response, since about 5% of those genes exhibit changes in transcription start sites. My work points to several levels at which such changes could be functionally important. For example, cases are found in which the location in the cell of the protein encoded by the gene may change, and other cases point strongly to importance for the efficiency of protein synthesis from the mature messenger RNA resulting from gene transcription and subsequent processing. Finally, my work has led to a new discovery of how the genetic reprogramming works during the activation of the immune response: My experimental design with investigation of gene activity very early after immune stimulation showed the induction of an early wave of hitherto overlooked regulatory factors. These regulatory factors have the potential to activate many of the genes known to be characteristic of the immune state. Thus, my work also lays the foundation for further investigations of transcriptional reprogramming during the activation of the plant immune response.