Intron sequences that stimulate gene expression in Arabidopsis
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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. Previously we used the IMEter algorithm in two independent ways to identify two motifs (CGATT and TTNGATYTG) that are candidates for involvement in IME in Arabidopsis. Here we show that both motifs are sufficient to increase expression. An intron that has little influence on expression was converted into one that increased mRNA accumulation 24-fold and reporter enzyme activity 40-fold relative to the intronless control by introducing 11 copies of the more active TTNGATYTG motif. This degree of stimulation is twice as large as that of the strongest of 15 natural introns previously tested in the same reporter gene. Even though the CGATT and TTNGATYTG motifs each increased expression, and CGATT matches the NGATY core of the longer motif, combining the motifs to make TTCGATTTG reduced the stimulating ability of the TTNGATYTG motif. Additional substitutions were used to test the contribution to IME of other residues in the TTNGATYTG motif. The verification that these motifs are active in IME will improve our ability to predict the stimulating ability of introns, to engineer any intron to increase expression to a desired level, and to explore the mechanism of IME by seeking factors that might interact with these sequences.
KeywordsIntron Gene expression Cis-element Motif Arabidopsis
We thank Dr. Neil Willits for statistical analysis, and Dr. Lesilee Rose and Jenna Gallegos for helpful comments on the manuscript. This work was supported in part by the United States Department of Agriculture, Grant Number 2006-35301-17072.
A.R designed and carried out most of the experiments, and wrote the manuscript. A.C. and N.K. assisted in the experiments. I.K. provided bioinformatic guidance in choosing motif mutations to test.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Rose AB, Emami S, Bradnam K, Korf I (2011) Evidence for a DNA-based mechanism of intron-mediated enhancement front. Plant Sci 2:98Google Scholar