An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana.

Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions.

Introns as Gene Regulators: A Brick on the Accelerator.

A picture is beginning to emerge from a variety of organisms that for a subset of genes, the most important sequences that regulate expression are situated not in the promoter but rather are located within introns in the first kilobase of transcribed sequences. The actual sequences involved are difficult to identify either by sequence comparisons or by deletion analysis because they are dispersed, additive, and poorly conserved. However, expression-controlling introns can be identified computationally in species with relatively small introns, based on genome-wide differences in oligomer composition between promoter-proximal and distal introns.

Intron DNA Sequences Can Be More Important Than the Proximal Promoter in Determining the Site of Transcript Initiation.

To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included.

Intron sequences that stimulate gene expression in Arabidopsis.

Related motifs strongly increase gene expression when added to an intron located in coding sequences. Many introns greatly increase gene expression through a mechanism that remains elusive. An obstacle to understanding intron-mediated enhancement (IME) has been the difficulty of locating the specific intron sequences responsible for boosting expression because they are redundant, dispersed, and degenerate.

The enduring mystery of intron-mediated enhancement.

Within two years of their discovery in 1977, introns were found to have a positive effect on gene expression. Numerous examples of stimulatory introns have been described since then in very diverse organisms, including plants. In some cases, the mechanism through which the intron affects expression is readily understood.

The effects of a stimulating intron on the expression of heterologous genes in Arabidopsis thaliana.

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated.

Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants.

Introns in a wide range of organisms including plants, animals and fungi are able to increase the expression of the gene that they are contained in. This process of intron-mediated enhancement (IME) is most thoroughly studied in Arabidopsis thaliana, where it has been shown that enhancing introns are typically located near the promoter and are compositionally distinct from downstream introns. In this study, we perform a comprehensive comparative analysis of several sequenced plant genomes.

Evidence for a DNA-Based Mechanism of Intron-Mediated Enhancement.

Many introns significantly increase gene expression through a process termed intron-mediated enhancement (IME). Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement.

Applying word-based algorithms: the IMEter.

Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression.

Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression.

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression.

Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events.

The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis.

Introns are often required for full expression of genes in organisms as diverse as plants, insects, nematodes, yeast, and mammals. To explore the potential mechanisms of intron-mediated enhancement in Arabidopsis thaliana, the effect of varying the position of an intron was determined using a series of reporter gene fusions between TRYPTOPHAN BIOSYNTHESIS1 (TRP1) and GUS. Two introns that differ in the degree to which they stimulate expression were individually tested at six locations within coding sequences and two positions in the 3'-UTR.

Requirements for intron-mediated enhancement of gene expression in Arabidopsis.

To explore possible mechanisms of intron-mediated enhancement of gene expression, the features of PAT1 intron 1 required to elevate mRNA accumulation were systematically tested in transgenic Arabidopsis. This intron is remarkably resilient, retaining some ability to increase mRNA accumulation when splicing was prevented by mutation of 5' and 3' splice sites, branchpoint sequences, or when intron U-richness was reduced. Enhancement was abolished by simultaneously eliminating branchpoints and the 5' splice site, structures involved in the first two steps of spliceosome assembly.