Kozak sequence acts as a negative regulator forde novotranscription initiation of newborn coding sequences in the plant genome

Abstract

The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ~100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

De novoactivated transcription of inserted foreign coding sequences is inheritable in the plant genome

“…As shown in Figure 2f, the relative abundances of the higher and lower fractions in T2-plants exhibited a greater bipolarization than they did in cultured cells; LUC transcription became much stronger in the Genic Sense and AS types, while it became much weaker in the Intergenic type (Figure 2f). As the Genic Sense and AS types were transcribed presumably by trapping the transcriptional activities of endogenous genes (Hata et al , 2020), these features suggested that gene-trapping events are more prone to occur in plants vs. cultured cells. Conversely, a type of transcriptional repression might have occurred on the Intergenic type in the T2 generation.…”

“…( a ) Breakdown of LUC insertion loci in T2-plants (upper panel) and T87 cells (lower panel) (Satoh and Hata et al , 2020) according to their transcription status compared with that of the inherent locus from WT transcriptome data. ( b ) Relative fractions of the breakdown classifications shown in (a) normalized to the percentage of untranscribed types in WT as 100%.…”

“…To examine the mechanism via which new genes acquire their promoters in the plant genome, we previously carried out an artificial evolutionary experiment (Satoh and Hata et al , 2020). To mimic a gene-birth event, we introduced exogenous promoterless coding sequences into Arabidopsis thaliana T87 cultured cells and analysed their transcriptional fates at the genome-wide level (Satoh and Hata et al , 2020). Interestingly, we found that promoterless coding sequences became transcriptionally activated via two distinct mechanisms: (1) the so-called promoter trapping, in which integrated sequences capture the endogenous promoter activities of pre-existing genes/transcripts; or (2) de novo transcriptional activation, which occurs ubiquitously across the entire genome, and does so stochastically in about 30% of the integration events independent of the chromosomal locus (Satoh and Hata et al , 2020).…”

“…To mimic a gene-birth event, we introduced exogenous promoterless coding sequences into Arabidopsis thaliana T87 cultured cells and analysed their transcriptional fates at the genome-wide level (Satoh and Hata et al , 2020). Interestingly, we found that promoterless coding sequences became transcriptionally activated via two distinct mechanisms: (1) the so-called promoter trapping, in which integrated sequences capture the endogenous promoter activities of pre-existing genes/transcripts; or (2) de novo transcriptional activation, which occurs ubiquitously across the entire genome, and does so stochastically in about 30% of the integration events independent of the chromosomal locus (Satoh and Hata et al , 2020). We speculated that the insertion of exogenous coding sequences might activate local chromatin remodelling to shape the promoter-like epigenetic landscape, resulting in such de novo transcriptional activation.…”

“…In this study, we aimed to establish a model system to elucidate the mechanism via which newborn coding sequences acquire their promoters and become fixed as functional genes in the plant genome. We carried out a large-scale promoter-trap screening in the T2 generation of A. thaliana plants under an experimental scheme similar to that used in our previous study of cultured cells (Satoh and Hata et al , 2020). By comparing the results obtained in plants with those of cultured cells, we concluded that de novo transcriptional activation together with chromatin remodelling is an inheritable phenomenon in the plant genome.…”