Most genes in eukaryotes (well, at least eukaryotes that are not Saccharomyces cerevisiae) possess introns, sequences that are transcribed by RNA polymerase II and subsequently spliced out from the primary transcript. Introns have been the subject of tremendous interest since their discovery in the 1970’s, and have provided much insight (and grist for controversy) into subjects as disparate as junk DNA, the RNA World, and mechanisms of gene expression. Among the still-unresolved matters today has to do with the timing of splicing – is it cotranscriptional* or does it occur after polII has released the transcript.
The case for co-transcriptional splicing has been built in part through numerous studies that reveal physical connections between splicing factors and the transcriptional complex; many (most) of these involve the so-called CTD (C-Terminal Domain) of RNA polymerase II. (This recent review summarizes this emerging field.) The general idea is that, owing to the association of splicing factors with the CTD of polII, they are able to bind the nascent transcript and initiate splicing before polII has completed the synthesis of the primary transcript.
It is with this backdrop that some recent developments in the area of gene expression in plants are of interest. Some 12 years ago, Alan Rose noted that introns can increase the expression of transgenes in plants. Subsequent studies (see the list of references at the end of this essay) revealed that this was not a general feature of introns, but rather was a property of a particular class of intron. Moreover, it turns out that the intron need not be spliced in order for enhancement to be seen, since introns crippled via alteration of splice junctions can still enhance expression. An enhancing intron “works” if it is near the 5′ end of the transcript and in its “normal” orientation; this latter characteristic distinguishes an enhancing intron from a transcriptional enhancer element. Modest (e.g., point) mutations do not seem to affect enhancement, but large changes in intron sequence can reduce the effect; this implicates intronic sequences but also indicates that a single, highly-specific sequence motif is not responsible for enhancement. Rose and his coworkers found that 5′-proximal introns have a different sequence profile than other introns in Arabidopsis. Using this, these authors identified a candidate enhancing motif and developed an algorithm by which enhancing introns may be identified.
So what is going on? How can an intron enhance expression seemingly independent of splicing and without possing a formal enhancer element? We really don’t know. As Rose states:
Introns can significantly affect gene expression in plants and many other eukaryotes in a variety of ways. Several types of gene regulation, both positive and negative, that involve plant introns are reviewed in this chapter. Some introns contain enhancer elements or alternative promoters, while many others elevate mRNA accumulation by a different process that has been named intron-mediated enhancement (IME). The introns involved in IME must be within transcribed sequences near the start of a gene and in their natural orientation to increase expression. The intron sequences involved are still poorly defined, and the mechanism of IME remains mysterious. A model of IME is presented in which introns increase transcript elongation.
The last sentence is intriguing, in that it suggests that the link between transcription and splicing may be behind intron enhancement. Recent studies (see the reading list at the end of the essay) that reveal a possible connection between RNA processing and chromatin modification may also be relevant to this problem.
Finally, it is important to know the scope of the phenomenon – is it seen only in Arabidopsis, or is it a more widely-conserved feature of gene expression? Along these lines, Rose et al. noted that:
The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.
Apropos of this, Bartlett et al. have documented that this phenomenon can be used to enhance expression of foreign genes in barley. Thus, along with being an interesting tool to further probe the conenctions between transcription and RNA processing, this phenomenon would appear to have some immediate utility in plant biotechnology.
A recommended reading list:
Recent reviews on the subject of links between transcription and RNA processing:
Egloff S, Murphy S. 2008. Cracking the RNA polymerase II CTD code. Trends Genet. 24, 280-288.
Pandit S, Wang D, Fu XD. 2008. Functional integration of transcriptional and RNA processing machineries. Curr Opin Cell Biol. 20, 260-265.
References related to cromatin modification and splicing:
Andersson R, Enroth S, Rada-Iglesias A, Wadelius C, Komorowski J. 2009. Nucleosomes are well positioned in exons and carry characteristic histone modifications. Genome Res 19, 1732-1741. (This is the one I link to in the essay; the following are also important)
Tilgner H, Nikolaou C, Althammer S, Sammeth M, Beato M, Valcárcel J, Guigó R. 2009. Nucleosome positioning as a determinant of exon recognition. Nat Struct Mol Biol.
16, 996-1001.
Kolasinska-Zwierz P, Down T, Latorre I, Liu T, Liu XS, Ahringer J. 2009. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat Genet. 41, 376-81.
Schwartz S, Meshorer E, Ast G. 2009. Chromatin organization marks exon-intron structure. Nat Struct Mol Biol. 16, 990-5.
References related to intron enhancement:
Rose AB, Last RL. 1997. Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1. Plant J. 11, 455-464.
Rose AB. 2002. Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA 8, 1444-1453.
Rose AB. 2004. The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J. 40, 744-751.
Rose AB, Elfersi T, Parra G, Korf I. 2008. Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell 20, 543-551
Rose AB. 2008. Intron-mediated regulation of gene expression. Curr Top Microbiol Immunol. 2008;326:277-90. (This is a review that is not available online.)
Bartlett JG, Snape JW, Harwood WA. 2009. Intron-mediated enhancement as a method for increasing transgene expression levels in barley. Plant Biotechnol. J., published online September 28, 2009.
* – cotranscriptional indicates a process that occurs while the nacsent transcript is still attached to, and being transcribed by, RNA polymerase II.
The RNA Underworld 