Identification of cis-regulatory motifs in first introns and the prediction of intron-mediated enhancement of gene expression in Arabidopsis thaliana

Abstract

Background Intron mediated enhancement (IME) is the potential of introns to enhance the expression of its respective gene. This essential function of introns has been observed in a wide range of species, including fungi, plants, and animals. However, the mechanisms underlying the enhancement are as of yet poorly understood. The goal of this study was to identify potential IME-related sequence motifs and genomic features in first introns of genes in Arabidopsis thaliana. Results Based on the rationale that functional sequence motifs are evolutionarily conserved, we exploited the deep sequencing information available for Arabidopsis thaliana, covering more than one thousand Arabidopsis accessions, and identified 81 candidate hexamer motifs with increased conservation across all accessions that also exhibit positional occurrence preferences. Of those, 71 were found associated with increased correlation of gene expression of genes harboring them, suggesting a cis-regulatory role. Filtering further for effect on gene expression correlation yielded a set of 16 hexamer motifs, corresponding to five consensus motifs. While all five motifs represent new motif definitions, two are similar to the two previously reported IME-motifs, whereas three are altogether novel. Both consensus and hexamer motifs were found associated with higher expression of alleles harboring them as compared to alleles containing mutated motif variants as found in naturally occurring Arabidopsis accessions. To identify additional IME-related genomic features, Random Forest models were trained for the classification of gene expression level based on an array of sequence-related features. The results indicate that introns contain information with regard to gene expression level and suggest sequence-compositional features as most informative, while position-related features, thought to be of central importance before, were found with lower than expected relevance. Conclusions Exploiting deep sequencing and broad gene expression information and on a genome-wide scale, this study confirmed the regulatory role on first-introns, characterized their intra-species conservation, and identified a set of novel sequence motifs located in first introns of genes in the genome of the plant Arabidopsis thaliana that may play a role in inducing high and correlated gene expression of the genes harboring them.

Exploring the Distribution of Single Nucleotide Polymorphisms Across Human Exons and Introns

“…In the aforementioned study of Back and Walther (8) that used Arabidopsis thaliana as the model genome, a high positive correlation was estimated between the sequence variation in the first exons and gene expression, which would explain higher genomic variability of the first exon observed in our study. Also, in a slightly different context of across-species comparison based on the reference genome sequence, Castle (4) observed higher variability of coding regions in the proximity of the start and stop codons, so the ones typically corresponding to the first and the last exons, that is in line with our observation of SNP excess in the first and the last exons.…”

“…These specific roles may explain why the human DNA’s first intronic sequence is considered the longest and highly dense of regulatory chromatin marks (24, 25) Considering the criterion of SNP density, the above-mentioned studies identified the first introns as the most conserved regions, that however, provided a highly non-uniform distribution of SNPs along the intronic sequence (26) does not directly translate to the total low number of SNPs. Interestingly, in Arabidopsis thaliana , Back and Walther (8) observed that the first introns harbour more SNPs than the subsequent introns.…”

“…But even within functional genomic units with a sequential structure, such as introns and exons, the density of SNPs is highly non-uniform (4), with clusters of adjacent SNPs being an often-observed characteristic of the human genome. In particular, Hodgkinson and Eyre-Walker (5) and Prendergast et al (6) estimated an excess of intronic SNPs located in a single-bp proximity to each other Matsushita and Kano-Sueoka (7) recently reported differential clustering of synonymous and non-synonymous SNPs among consecutive exons of the human HLA-A gene, while in the whole genome scope Back and Walther (8) observed in Arabidopsis thaliana a higher density of SNPs located in the first intron than in the next introns. It has been widely agreed in the literature that such non-random distribution of SNPs must have evolutionary impactions and is the result of mutational hotspots, or that SNP clusters arise due to structural properties of DNA that mechanically promote the accumulation of such point mutations.…”

Genomic imprints of unparalleled growth

“…40–50‐fold slower and the IPG is about one third of that of Ohadii , isolated from the harsh desert crusts environment, where the limited daily time interval enabling photoautotrophic activity favors fast growth. This is consistent with the suggestion that the presence of introns accelerates the expression of genes without serving as a binding site for transcription regulators, a phenomenon termed ‘intron‐mediated enhancement’ (Shaul, 2017; Lasin et al ., 2020; Back & Walther, 2021; and references therein). Furthermore, a recent study showed that in addition to the first intron, those located downstream also affect the expression of rbcS (the nuclear gene encoding the small subunit of RuBisCO) in C. reinhardtii (Baier et al ., 2018).…”

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

“…For example, the first intron of maize ubiquitin 1 ( ZmUbi1 ) located in the 5′-UTR region combines with CaMV 35S promoter leads to a over 90-fold increasement of gene expression in maize and bluegrass ( Vain et al, 1996 ). Many works have attempted to identify the key cis-elements in this process, but the detailed mechanism is still not clear, since it has been found that the sequence and splicing process are not the key feathers of these introns ( Rose and Beliakoff, 2000 ; Rose, 2004 ; Back and Walther, 2021 ). Thus, it’s promising to engineer these introns to enhance the strength of the promoters that drive the expression of the CRISPR/Cas9 system.…”