Kozak Sequence Acts as a Negative Regulator for De Novo Transcription 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 novo activated transcription of inserted foreign coding sequences is inheritable in the plant genome

“…Previously, based on the cultured cell experiment, we found that exogenously inserted promoterless genes became transcriptionally activated in two distinct types: promoter trapping and de novo transcriptional activation [ 26 , 27 ]. To examine whether similar transcriptional activation mechanisms occurred in our T2-plants, the abundance of the transcribed fraction was compared between the corresponding insertion types of T2-plants and cultured cells ( Fig 2D and 2E ).…”

“…As shown in Fig 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 ( Fig 2F ). As the Genic Sense and AS types were transcribed presumably by trapping the transcriptional activities of endogenous genes [ 27 ], these features suggested that gene-trapping events are more prone to occur in plants than in cultured cells. Conversely, a type of transcriptional repression might have occurred on the Intergenic type in the T2 generation.…”

“…In the T2:161 line, the TSS was located on the 3′ end of an endogenous gene (AT3G23750), where no detectable transcripts existed in the WT genome ( Fig 5A ). It is plausible to propose that this was caused by activating (rather than trapping) a cryptic antisense transcript of the given locus [ 27 ]. Conversely, we speculated that the T2:205 line may be transcribed from a de novo -activated TSS located in the proximal intergenic region, although we could not identify this TSS in this study.…”

“…Conversely, we speculated that the T2:205 line may be transcribed from a de novo -activated TSS located in the proximal intergenic region, although we could not identify this TSS in this study. This speculation was based on a previous finding from the cultured cell experiment: de novo TSS occurs about 100 bp upstream of the inserted coding sequences in the intergenic region [ 27 ]. The localization pattern of H2A.Z in the T2:205 line agreed with this prediction, as the H2A.Z signal clearly dropped to almost zero at 200 bp upstream of the LUC insert ( Fig 4E ).…”

“…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 independently of the chromosomal locus [ 26 ]. The transcription start sites (TSSs) analysis revealed that the de novo transcription occurred from the 5’ close proximal region of the inserted foreign coding sequences [ 27 ]. We speculated that the insertion of exogenous coding sequences might activate local chromatin remodelling to shape the promoter-like chromatin configuration, resulting in such de novo transcriptional activation in the plant genome.…”