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Plant Science 237 (2015) 8–15

Contents lists available at ScienceDirect

Plant Science

j ourna l ho me pa g e: www.elsev ier .com/ locate /p lantsc i

eview

he enduring mystery of intron-mediated enhancement

enna E. Gallegos, Alan B. Rose ∗

epartment of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA, USA

r t i c l e i n f o

rticle history:eceived 12 March 2015eceived in revised form 21 April 2015ccepted 22 April 2015vailable online 30 April 2015

eywords:ntron-mediated enhancementMEterene expression

a b s t r a c t

Within two years of their discovery in 1977, introns were found to have a positive effect on geneexpression. Numerous examples of stimulatory introns have been described since then in very diverseorganisms, including plants. In some cases, the mechanism through which the intron affects expression isreadily understood. However, many introns that affect expression increase mRNA accumulation throughan unknown mechanism, referred to as intron-mediated enhancement (IME). Despite several decades ofresearch into IME, and the clear benefits of using introns to increase transgene expression, little progresshas been made in understanding the mechanism of IME. Several fundamental questions regarding therole of transcription and splicing, the sequences responsible for IME, the involvement of other factors,and the relationship between introns and promoters remain unanswered. The more we learn about the

ranscriptionRNA accumulationene regulation

properties of stimulating introns, the clearer it becomes that the effects of introns are unfamiliar anddifficult to reconcile with conventional views of how transcription is controlled. We hypothesize thatintrons increase transcript initiation upstream of themselves by creating a localized region of accessiblechromatin. Introns might represent a novel kind of downstream regulatory element for genes transcribedby RNA polymerase II.

© 2015 Published by Elsevier Ireland Ltd.

ontents

1. An introduction to the characterization and exploration of IME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.1. Defining IME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2. Impediments to molecular analysis of IME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3. Computational analysis of IME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2. Unsolved mysteries of IME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.1. Is splicing necessary for IME?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.2. Is IME caused by sequences in the RNA, the DNA, or both? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.3. What intron sequences are responsible for IME? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.4. Does IME involve sequence-specific interactions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.5. What stage of gene expression is most affected by introns? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

3. Models of IME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.1. Previously published models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123.2. A new model of IME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: IME, intron-mediated enhancement.∗ Corresponding author. Tel.: +1 530 754 9892; fax: +1 530 752 3085.

E-mail addresses: jegallegos@ucdavis.edu (J.E. Gallegos), abrose@ucdavis.eduA.B. Rose).

ttp://dx.doi.org/10.1016/j.plantsci.2015.04.017168-9452/© 2015 Published by Elsevier Ireland Ltd.

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1. An introduction to the characterization and explorationof IME

Introns are easy to overlook as important gene regulatory ele-ments, as illustrated by their inclusion for many years in thecategory of “junk DNA.” However, the list of introns shown to sig-nificantly boost gene expression in plants and other organisms

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ontinues to grow. Very little is known about the mechanismshrough which introns increase expression. Much of what we donow about introns is difficult to fit into the idea that the level ofranscript derived from a gene is mostly controlled by the recruit-

ent of the transcription machinery by general and regulatoryranscription factors binding to specific DNA sequences in its pro-

oter. We will use the term ‘promoter’ in the narrow sense ashe sequences around and upstream of the start of transcription,ather than the broader definition of promoters as any sequencesincluding introns) that influence the expression of a gene. Theole of introns must be reconciled with the large body of exist-ng knowledge of promoter function, although there is still plentyf room for new insights into how genes are expressed. In thiseview, we will highlight some of the unresolved issues in the field,nd speculate on the ways in which introns might bolster mRNAccumulation.

.1. Defining IME

The term intron mediated enhancement (IME) has been used asn umbrella to include any case in which an intron-containing con-truct is expressed more highly than an intron-less counterpart. Theffect of an intron is usually quantified by dividing the amount ofRNA accumulation or reporter enzyme activity produced from an

ntron-containing gene by the amount made by an intron-less con-rol. Some genes are absolutely intron-dependent [1,2], in that evenith a fully intact promoter they are expressed at undetectably

ow levels unless they contain an intron, in which case this ratios essentially infinite. The effects of introns on mRNA levels cane quite large, depending on the intron and the gene, and intron-aused increases of ten-fold or more in expression levels are ofteneported [3–5]. There do not appear to be specific relationshipsetween introns and promoters since many different stimulating

ntrons can boost expression from the same promoter [6], andhe same stimulating intron can increase the expression of mul-iple genes [2,3]. Many different effects of introns have been found,lthough the mechanisms responsible for differences in expressionften are not investigated. Examples of introns that house canonicalnhancer elements or alternative promoters have been described7–9] and the known interactions between splicing factors andNA polymerase II provide multiple opportunities for introns toffect transcript initiation, capping, elongation, and 3′ end forma-ion [10,11]. In addition, the proteins of the exon junction complexhat are deposited on the mRNA during splicing can increase thefficiency of nuclear export and translation [12,13]. While intronsave been shown to affect various stages of gene expression, in thiseview, IME will specifically refer to the effects of an intron withhe following three characteristics: 1) The intron increases mRNAccumulation; 2) large portions of the intron can be deleted withoutliminating its effect on expression; and 3) the intron only increasesRNA levels when downstream of, and close to, the start of tran-

cription. Plant introns that fit this narrow definition include thoserom the Adh1 and Sh1 genes of maize [3,4,14], the OsTubA1 genef rice [15], and the PRF2, TRP1, UBQ10, and AtMHX1 genes of Ara-idopsis [6,16–21]. Several other introns possess two of the threeriteria and will likely join the club when the missing experimentsre performed.

These constraints rule out most of the well-understood effectsust mentioned as contenders for the main basis of IME, such asnhancers or the beneficial effects of the exon junction complex.he act of splicing cannot be the cause of IME, because somefficiently spliced introns make no difference to mRNA accumu-

ation [6]. In essence, the mystery of IME is this: how can onlyome introns have such a large effect on mRNA levels from down-tream of the transcription start site if they do not contain annhancer?

cience 237 (2015) 8–15 9

1.2. Impediments to molecular analysis of IME

Despite nearly three decades of IME research in plants, and theobvious practical benefits of increasing expression in biotechnol-ogy applications, our knowledge of how introns affect mRNA levelsremains surprisingly rudimentary. One reason progress in under-standing IME has been slow is that the numerous interconnectionsbetween the various steps of gene expression make it difficult toisolate a single variable. Seemingly simple questions such as “issplicing required for IME?” are not easy to address even thoughsplicing can be eliminated with a single nucleotide change. Theintron sequences that are then retained in the mRNA will havemultiple cascading effects that differ depending on the locationof the intron. These could include changing the length and struc-ture of the 5′-UTR and thus translation efficiency and possiblymRNA stability, introducing a new initiation codon, terminatinga reading frame and triggering nonsense-mediated mRNA decay,preventing deposition of the exon junction complex, or altering thestructure and activity of the reporter enzyme. Another factor thathas hampered IME research has been the difficulty of generatingaccurate quantitative data. Transient assays are notoriously vari-able between experiments and present technical challenges for athorough molecular analysis of expression. Furthermore, the effectof an intron in transient expression assays is an order of magni-tude smaller than in stably transformed lines [22,23]. The use oftransgenic plants requires an unpleasant choice between dealingwith large line-to-line variation in expression due to differences intransgene copy number, or the laborious and rate-limiting task ofidentifying single-copy lines. Furthermore, knowledge gained fromone intron may or may not be applicable to others. Either a verysmall number of introns can be studied in enough depth to com-pare characteristics, or more can be examined superficially withoutbeing sure that all affect expression by the same mechanism. TheIME literature is rich with examples of introns that were discoveredto boost expression, but very few labs have made the commitmentrequired to investigate the mechanisms at work. These constraintshave limited the number of introns in which IME has been unam-biguously demonstrated.

1.3. Computational analysis of IME

What proportion of introns exhibit IME? The publication recordis likely to be an unreliable indicator because introns that signifi-cantly boost expression are more newsworthy than those that haveno effect. Addressing this question computationally and on a broadscale was made possible by the development of the IMEter algo-rithm [24]. The IMEter is an important tool because it providesa means of predicting which introns and sequences are likely toaffect mRNA levels. This information can then be used to search forconserved features among stimulating introns and the genes thatencode them. The IMEter is based on the observation that intronsthat increase mRNA accumulation from the 5′ end of a gene usu-ally have no effect from the 3′ end [3,16,25]. More specifically,introns lose their ability to exhibit IME approximately one kilo-base from the start of transcription [17]. This led to the findingthat promoter-proximal introns as a group differ structurally fromother introns, as measured by the frequencies of all possible k-mers (nucleotide sequences of a given length k) in the populationsof promoter-proximal and distal introns. The IMEter calculates ascore that expresses the degree to which the k-mer composition of agiven intron is similar to promoter-proximal introns genome-wide[24]. The conclusion that the location-based difference in intron

composition is related to IME is supported by the strong correla-tion between the IMEter score and stimulating ability of introns(Fig. 1B, [24]). Around 5% of Arabidopsis introns would be expectedto increase mRNA accumulation from the TRP1:GUS reporter 5-fold

10 J.E. Gallegos, A.B. Rose / Plant Science 237 (2015) 8–15

Fig. 1. (A) IMEter 2.0 scores of all introns in the Arabidopsis genome individually plotted against their distance from the start of transcription. Approximately 5% of introns havescores of 20 or more, and virtually all of these are located within 1 kb of the transcription start site. (B) Correlation between intron IMEter scores and effect on expression. Forall Arabidopsis introns whose effect on TRP1:GUS expression has been measured in single-copy transgenic lines, the degree to which the intron increased mRNA accumulationis plotted against its IMEter 2.0 score. Error bars indicate standard deviations. (C) IMEter score distributions are conserved in plants. IMEters were generated for each indicatedspecies and used to calculate average IMEter 2.0 scores of introns separated by their distance from the start of transcription into 200 nt bins.

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r more based on their IMEter scores (Fig. 1A). Because the fre-uency of high-scoring introns is three times higher in first intronshan in the population of all introns, the expression of perhaps 15%f the genes in Arabidopsis could be influenced by IME. IMEtersave been developed for virtually all of the plants whose genomeequences are available in the Phytozome database (http://korflab.cdavis.edu/cgi-bin/IMEter 2014/web-imeter2.1.pl). All but a fewlgae also exhibit compositional differences in their promoter-roximal introns ([20], Keith Bradnam, personal communication).he distribution of IMEter scores in higher plant species is similaro that in Arabidopsis (Fig. 1C), suggesting that the Arabidopsis fig-res are reasonable rough estimates for the number of stimulating

ntrons in most plants.

. Unsolved mysteries of IME

The fundamental questions about the mechanism of IME thatemain open provide a measure of the depth of our ignorance abouthat stimulating introns are doing. These include the following:oes an intron need to be spliced to increase mRNA accumulation?

s IME primarily caused by the intron sequences in the DNA that iseing transcribed, the RNA that is produced, or both? What intronequences are responsible for IME? Does IME involve sequence-pecific interactions between protein(s) and nucleic acids? Whattage of gene expression is most affected by introns? Progress iseing made on some of these basic issues but others remain stuck

n the starting gate.

.1. Is splicing necessary for IME?

For reasons described above, addressing the need for splicing inME is not a simple task. The most serious concern, especially whenxpression is being measured as enzyme activity, is ensuring thathe intron sequences retained in the mRNA when splicing is pre-ented do not disrupt the reading frame of the reporter gene. Tovoid this problem, three groups have tested the effects of blockingplicing using either an intron that lacks start codons located in the′-UTR, or an intron with no in-frame stop codons positioned in cod-

ng sequences. Unfortunately, the divergent results obtained didot clarify the need for splicing in IME. The 1028 nt first intron of theaize Sh1 gene, and a 145 nt deletion derivative of the intron, each

nhance transient expression from the 5′-UTR of a CaMV35S:CATusion construct 20–50 fold in maize protoplasts [4]. When splic-ng of the deletion derivative is prevented by mutations at bothplice sites, the mutated intron stimulates expression just 2 fold4]. Expression was determined by enzyme activity but mRNA lev-ls were not measured in these experiments. Similarly, when bothplice sites of the intron in the 5′-UTR of the AtMHX gene wereutated in an AtMHX:GUS fusion in transgenic Arabidopsis, the

timulating ability of the intron was decreased from 272-fold to 5-old at the level of GUS enzyme activity, with a comparable decreasen mRNA levels [18]. The finding that the stimulation caused by twoifferent introns largely disappears when splicing is prevented sug-ests a role for the splicing machinery in IME. In contrast, the 5-foldncrease in mRNA accumulation caused by the TRP1 first intron inoding sequences of a TRP1:GUS fusion in transgenic Arabidopsisas only slightly reduced by mutations in the 5′ splice site, elimi-ating all possible branchpoint sequences, or making the intron toomall to be spliced [6,19], suggesting that splicing is not necessaryor IME. The inability of some efficiently spliced introns to affect

RNA levels shows that splicing clearly is not sufficient for IME.

How can these disparate results be reconciled? One previously

uggested possibility [18] is that there are two separate effects onRNA levels, a splicing-independent increase of 2- to 5-fold, and a

arger splicing-dependent mechanism that is present in only some

cience 237 (2015) 8–15 11

introns. The splicing requirements for IME could vary if the introns,genes, and organisms studied are simply different in other unrecog-nized ways. Finally, the locations of the introns might be significant,as retained introns in the 5′-UTR would have a greater effect onthe initiation of translation than those in coding sequences. There-fore, differences in enzymatic activity observed when splicing of anintron in the 5′ UTR is prevented may be due to effects on transla-tion rather than mRNA production. The question of whether or notsplicing is required for IME could be settled if intron sequences arefound to continue to boost mRNA levels when extracted from anintron and placed in an exon.

2.2. Is IME caused by sequences in the RNA, the DNA, or both?

The inability of introns to exhibit IME from outside of tran-scribed sequences, and the possible splicing requirement, suggestthat the mechanism of IME depends on intronic RNA. However,the need to be transcribed does not necessarily mean that IMEis caused by intron sequences present in the RNA; the samesequences also exist in the DNA and could influence the tran-scription machinery from there. Recent evidence supporting aDNA-based model includes the observation that, in the context ofa spliceable intron, sequences from stimulating introns increaseexpression equally well in both orientations [26]. This would seemto eliminate RNA-based models because inverting the intron frag-ment would drastically change the sequence of the RNA into itsreverse complement and therefore significantly disrupt any cis-acting RNA sequences involved in IME. The inverted fragmentstested do not contain any obvious palindromes or secondary struc-tures that would be the same in both orientations, but the existenceof subtle conserved RNA structures cannot be ruled out. Additionalsupport for a DNA-based mechanism includes the finding that IME-ter scores more accurately reflect the stimulating ability of an intronwhen calculated using both strands rather than only the sensestrand of the intron [26]. However, roles in IME for intronic RNAsequences or the splicing machinery remain possible. For exam-ple, R-loops formed between the nascent transcript and the DNAtemplate would be impervious to changes in the orientation ofsequences and could influence the rate of transcription. While thequestion remains open, an IME function seems probable for theintron sequences in the DNA.

2.3. What intron sequences are responsible for IME?

The fact that efficiently spliced introns widely differ in theireffect on mRNA levels indicates that some contain more, or moreactive, stimulating sequences than others. Identifying these stimu-latory sequences has been difficult because large internal deletionstypically do not reduce the activity of stimulatory introns, and theseintrons share no obviously conserved sequences. One reason inter-nal deletions can reduce, but do not eliminate, IME is that thestimulating sequences can be redundant and dispersed through-out the intron. For example, all four of the 54–113 nt sections thatcomprise the 304 nt UBQ10 intron increase mRNA accumulationwhen inserted into the non-stimulating COR15a intron [24]. Therelatively small number of introns with known effects on mRNAaccumulation, and the large diversity in length and sequence ofthose introns, preclude searching computationally for sequencesshared only by stimulating introns. It is also possible that IME iscaused by the physical properties of the nucleic acid rather than asequence-specific interaction with a protein, which may make theresponsible sequences less obviously conserved and thus more dif-

ficult to identify. In maize and Arabidopsis, T-richness rather thana specific sequence was suggested to be important for IME [4,6].In contrast, until recently the only other sequence to have beenimplicated in IME is a GC-rich octamer in maize [27]. The modest

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ffect on expression of these elements compared to entire intronsuggests that other sequences have larger roles in IME.

The IMEter has been used in two different ways to findequences involved in IME. In one, the sequence of the stimu-atory UBQ10 intron was scanned in a sliding window to locatehe sequences that make the largest contribution to the IMEtercore and presumably have the greatest effect on mRNA levels20]. Modifying the UBQ10 intron to eliminate the highest peaksf IMEter score cut the stimulating ability of this intron in half,trengthening the correlation between an intron’s IMEter score andts effect on expression. The majority of the peaks contained theequence CGATT, and adding 11 copies of this motif to the non-timulating COR15a intron converts it into one that increases mRNAccumulation more than 6-fold [20]. In an independent approach,otif-finding programs were used to search for sequences that are

ver-represented in introns with high IMEter scores. For exam-le, TTNGATYTG is more abundant in high-scoring Arabidopsis

ntrons than in low-scoring introns [24]. The functional signifi-ance of this motif is still being evaluated but preliminary resultsuggest that it is even more active than the CGATT sequence,hich matches the NGATY core of the longer motif. CGATT and

TNGATYTG are the first specific sequences to be tested thattrongly increase mRNA levels in a dose-dependent manner fromithin an intron in any system. Even though these sequences are

ufficient for IME, there must be other sequences capable of causingME, as not all stimulating introns contain a perfect match to either

otif.

.4. Does IME involve sequence-specific interactions?

The identification of sequences sufficient to drive IME provideshe first toehold for identifying factors that may interact with thems part of the mechanism of IME. The isolation of such a factorould constitute a major advance in understanding IME. How-

ver, the decision of whether to seek targets that bind to intronicNA or RNA is not the only peril that will be associated with a

earch for an IME factor. The redundant and dispersed nature ofhe sequences involved in IME, the apparent lack of highly con-erved sequences shared by stimulating introns, and the need forntrons to be downstream of the start of transcription to affect

RNA levels, suggest that conventional ideas of gene expressionontrol by proteinaceous transcription factors binding to conservedis-acting promoter sequences may not apply to IME. We shouldeep our minds open to alternative ideas. For example, IME coulde caused either by the physical structure of the DNA or RNA itself,r could involve sequence non-specific interactions between theucleic acid and proteins such as histones or hnRNPs.

.5. What stage of gene expression is most affected by introns?

Virtually all of the steps of mRNA biosynthesis and breakdownave been implicated as the primary level at which IME affectsRNA accumulation. The observation that a stimulating intron

nder the control of a tissue-specific promoter can cause strongxpression in tissues in which the promoter is normally inactivehows that introns can have a stronger effect than promoters onene expression and suggests that introns primarily affect tran-cript initiation [2,16,28]. A role for introns in increasing transcriptlongation would explain why introns boost mRNA accumulationnly when located in transcribed sequences near the promoter [17].n this scenario, the transcription machinery might have a low basalevel of processivity that is somehow increased by the intron if it

s encountered soon after initiation, thereby raising the probabil-ty of generating full-length stable transcripts. The small differencen signal obtained in nuclear run-on transcription assays of genes

ith and without an intron suggests that introns primarily increase

cience 237 (2015) 8–15

mRNA accumulation by a post-transcriptional mechanism [29].Transcripts from intron-containing genes are generally more sta-ble than those from intron-less genes [30], suggesting that intronscould increase mRNA levels by reducing mRNA decay. While intronscertainly could have more than one effect, some of these expla-nations are mutually incompatible. For example, introns cannotmainly increase transcript initiation and also predominantly act ata post-transcriptional level. We suspect that the previous failuresto detect an intron-associated increase in the rate of transcriptionmay be unreliable because they are negative results possibly causedby the technical difficulty of isolating nuclei and performing run-ontranscription assays in plants.

3. Models of IME

3.1. Previously published models

With so many basic questions unanswered, there is ample roomto speculate about possible ways for introns to increase mRNA lev-els. Indeed, numerous diverse models of IME have been proposed.Virtually all biochemical investigations of IME have been performedusing yeast or mammalian cells. The possibility that similar mech-anisms operate in plants remains to be evaluated. A speculativemodel is that introns boost expression by acting as molecular lubri-cants, dissipating the torsional strain generated by transcription[31]. Another DNA-based model for which there is experimentalsupport in yeast is that a stimulating intron causes DNA looping thatbrings the 5′ and 3′ ends of a gene into proximity, thereby favoringtranscription re-initiation [32]. Re-initiation is also the key elementof a model based on interactions between the U1 snRNA splicingfactor and the general transcription initiation factor TFIIH in HeLacells [33]. Interactions between human snRNP splicing factors andthe protein TAT-SF1 increase transcript elongation, rather than ini-tiation, by recruiting the transcription elongation factor pTEFb [34].In addition, interactions between components of the splicing andpolyadenylation machineries could lead to greater mRNA levelsby increasing the efficiency of polyadenylation of some transcripts[35].

3.2. A new model of IME

We offer the following model as a hypothesis for the main effectthat introns have on mRNA accumulation. We propose that intronsincrease transcript initiation or re-initiation within a limited zoneupstream of themselves. Evidence from other organisms supportsthe idea that introns can control initiation and transcription startsite selection in some cases. The ENCODE data from humans indi-cates that transcription often initiates a few hundred nucleotidesupstream of first introns in genes with long first exons [36]. In ourmodel, the need for introns to be near the beginning of a gene toincrease expression is based on the limited size of the initiation-favoring zone created by the intron, and the location of potentialtranslation start codons (Fig. 2). If, as proposed, transcription initi-ates upstream of the intron, and translation begins at the first ATGin an mRNA, there will be a relatively small range of intron loca-tions near the 5′ end of a gene in which the first start codon in theresulting mRNA will be that of the main open reading frame of thereporter gene (Fig. 2A and B). Transcripts that initiate outside ofthat window (Fig. 2C and D) will not produce a full-length proteinand may be unstable if the first ATG initiates a short open readingframe. This is because transcripts containing a stop codon upstream

of an intron or a long distance from the transcription terminationsite (creating a long 3′-UTR) can be subject to nonsense-mediatedmRNA decay [37]. The idea that introns can control transcript ini-tiation would explain why some genes are strictly dependent on

J.E. Gallegos, A.B. Rose / Plant S

Fig. 2. Model of intron-mediated enhancement. (A) Without an intron, transcriptsthat initiate at the normal start site in the promoter encode functional and stablemRNAs because the first ATG in the mRNA is the start codon for the full-lengthprotein. (B) An intron near the promoter causes additional transcription from sites,including the normal start, within a zone of influence (a chromatin structure thatfavors initiation) upstream of the intron. When the intron is too far downstream (C)or is moved upstream into the intergenic region (D), the first ATG in transcripts thatinitiate in the zone of influence is not the start codon of the main open reading frame.These transcripts are unstable because the first ATG in these mRNAs initiate a shortopen reading frame whose stop codon can be recognized as premature and therebytrigger nonsense-mediated mRNA decay. Rectangle, coding sequences of gene. Dia-monds, ATGs. Triangle, stimulating intron. Yellow cloud, intron zone of influence.AB

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rrows, transcription initiation sites. Red lines, transcripts from normal promoter.lue lines, transcripts caused by the intron. Dashed lines, unstable transcripts.

ntrons for expression, as these genes may rely on introns to drivenitiation and may not have an independently functioning con-entional promoter. For example, the unc-54 gene in C. elegansan function with its promoter deleted but not without its introns38]. The tissue-specific pattern of expression of a gene could be

uperseded by an intron because prior activity of a promoter is notequired if the intron determines initiation.

We further propose that the mechanism through which intronstimulate transcript initiation is by creating a favorable local

ig. 3. The distribution of average IMEter scores (A) mimics activating histone modificatio20] (A) and [43] (B), with permission.

cience 237 (2015) 8–15 13

chromatin structure (represented by a yellow cloud in Fig. 2).This favorable structure could be an open area relatively free ofnucleosomes, or a region of chromatin marked by activating his-tone modifications or the presence of an accessory protein thatrecruits the transcription machinery. This hypothesis is supportedby the striking similarity between the distribution of IMEter scoresand activating histone modifications in Arabidopsis (Fig. 3), and theenrichment of activating histone modifications at the 5′ boundariesof first introns in humans [36]. The stimulating sequences withinan intron could create the proposed activating chromatin structureeither by recruiting enzymes that place activating modificationson nearby histones, or by influencing nucleosome occupancy.Sequence composition affects DNA flexibility and can significantlyalter nucleosome binding preferences [39]. This may be the rea-son that introns tend to associate with fewer nucleosomes than doexons [40]. Stimulatory introns could accentuate these differencesin nucleosome occupancy or might have more abundant sequencefeatures such as polydT tracks [4], polypyrimidine stretches [41],or AT periodicity [42] that could influence initiation in other ways.The amount of chromatin that could be affected by an intron hasto include at least three nucleosomes because introns are capableof increasing mRNA accumulation from 550 nt downstream of thenormal start of transcription [17], and each nucleosome occupiesabout 150 nt of DNA.

Our model is not mutually exclusive with previous models ofIME described above. It differs from them in that transcript initi-ation is driven by sequences within specific introns, rather thangeneral splicing factors, and its ability to explain the promoterproximity requirement of IME. Our DNA-based model is not incon-sistent with the apparent splicing requirement for IME of someintrons. The many physical interactions between the splicing andtranscription machineries ensure that nascent transcripts are effi-ciently processed, and also provide opportunities for co-regulationof these activities. We speculate that the inability of the spliceo-some to process a mutated intron in the pre-mRNA could inhibittranscript elongation, even if IME signals in the intron DNA stimu-late transcript initiation. Alternatively, the architecture of DNA or

chromatin could be altered by feedback in a splicing-dependentmanner, as suggested in yeast [32].

ns (B) relative to transcription start sites in the Arabidopsis genome. Modified from

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4 J.E. Gallegos, A.B. Rose / P

. Concluding remarks

It is our hope that by highlighting the puzzling features of thishenomenon, more researchers will be sufficiently intrigued by

ME to tackle some of the unresolved issues. IME, as describedn this review, represents a subset of the ways in which intronsnfluence gene expression. Studies on the effects of introns onther processes such as translation are likely to generate addi-ional surprises [21]. The field of intron research would greatlyenefit from having more bright minds seeking ways to answerhe open questions. Simply increasing the number of character-zed introns would help to clarify some issues such as the need forplicing in IME, and improve the computational analysis of stimu-ating introns and the sequences they share. The idea that intronsffect chromatin structure can be tested by nuclease sensitiv-ty, chromatin immunoprecipitation experiments, and an analysisf existing data regarding chromatin structure near stimulatingntrons. An in vitro analysis of the effect of introns on transcrip-ion may require the use of a species more biochemically andenetically tractable than Arabidopsis or maize. The ideal organismill contain numerous modestly sized introns and be competent

or integrative transformation, such as the microalgae Ostreococ-us lucimarinus or the fission yeast Schizosaccharomyces pombe. Itill take some time to determine if the characteristics of IME in

new experimental system are similar to those found in vascularlants.

We believe that the unusual characteristics of IME and theery large effect that some introns have on mRNA produc-ion reflect a major unrecognized method of gene regulation.

hile regulatory elements can be located downstream of theranscription initiation sites of genes transcribed by RNA poly-

erases I and III, enhancers are the only downstream controllinglements currently recognized for genes transcribed by RNA poly-erase II. Even though investigating the mystery of IME has

roved challenging, the reward for solving the puzzle could ben understanding of an entirely new method of controlling genexpression.

cknowledgements

We thank Dr. Lesilee Rose and Derrick Hicks for helpful com-ents on the manuscript, and Dr. Keith Bradnam for computational

ssistance. The authors gratefully acknowledge research supportver the years from the United States Department of Agriculturend the National Science Foundation.

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