Progress 08/01/03 to 07/31/07
Outputs
The goal of my research is to understand how introns elevate gene expression in plants. Towards that end, the purpose of this project was to test the hypothesis that introns stimulate mRNA accumulation by increasing transcript elongation. The main approach used was to create a series of reporter genes, each with a single intron at one of eight different locations, and then to measure the ability of the intron to elevate expression from each position in single-copy transgenic Arabidopsis plants. This analysis revealed two distinct mechanisms through which introns affect expression. Introns boost mRNA accumulation the most when they are nearest to the start of the gene, and the effect on expression declines as the intron is moved down the gene until it disappears completely approximately one kilobase from the promoter. This same pattern was observed for two introns that differ in the degree to which they stimulate mRNA accumulation. The position dependence suggests that
introns act during transcription, and supports the hypothesis that introns somehow increase transcript elongation. When expression relative to the intronless control was measured as enzyme activity, the enhancement was approximately twice that seen as mRNA accumulation, suggesting that introns also may double translational yield (the amount of protein made per unit of mRNA). The apparent effect on translation was roughly two-fold regardless of intron location and showed very little variation between introns, indicating a mechanism distinct from that affecting mRNA accumulation. Biochemical evidence to support an effect of introns on transcript elongation was sought using chromatin immunoprecipitation, although personnel issues prevented completion of this second aim. The person hired to establish this promising but technically challenging procedure resigned before generating useful results, as did her replacement. The original proposal for this research contained a third aim, which
was dropped from the revised aims to reflect the amount of work considered feasible under the modified budget. However, significant progress was made on this aim, and the preliminary results obtained will be further pursued under the funding awarded when this grant was renewed. The goal of this aim was to localize the intron sequences responsible for enhancement by creating deletions and hybrids between two introns with very different effects on expression. These experiments clearly showed that each quarter of a strongly enhancing intron can be deleted without significantly reducing the ability of that intron to stimulate expression, and the first three of the quarters can each mediate enhancement when inserted into an intron that has little effect on expression. This demonstrates that enhancing sequences are distributed throughout most of the stimulatory intron, a finding that rules out many potential models of intron-mediated enhancement. All of the above results are meaningful
because they illustrate several characteristics of the mechanisms through which introns affect expression, which are unlike other known types of gene regulation.
Impacts
The unparalleled biosynthetic capabilities of plants can be harnessed to synthesize many valuable substances such as medicines, edible vaccines, or unusual oils. In most cases, success depends on making large amounts of a specific protein, and ways to increase gene expression are enormously beneficial. Most of the candidate genes for such practical applications will be from non-plant sources and will lack introns naturally (e.g. viral or bacterial genes), or their introns will have been removed because plants are poor at splicing foreign introns. The expression of these genes will be increased by the addition of a plant intron, and this research provides the knowledge necessary to choose or engineer an intron appropriate for the desired level of expression and to place introns where they will do the most good. In addition to these practical considerations, the study of introns will continue to increase our understanding of the fundamental events of gene expression.
Introns are intriguing because they can have a huge effect on gene expression, yet very little is known about how they operate except that it can't be by the same mechanisms as better known regulatory elements such as enhancers. Introns are another example of sequences once considered 'junk DNA' whose importance is becoming clearer.
Publications
- Rose, A.B. 2007. Book Review: Plant Gene Expression. Science STKE, pe26.
- Belostotsky, D.A. and A.B. Rose 2005. Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events. Trends in Plant Science. 10:347-353.
- Rose, A.B. 2004. The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant Journal 40:744-751.
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Progress 01/01/06 to 12/31/06
Outputs
The goal of my research is to understand how introns enhance gene expression in plants. The aims of this proposal were to determine the effect on expression of varying intron position in coding sequences and to test for intron-mediated changes in RNA polymerase II elongation and phosphorylation. The first aim has been completed and the results published. The main finding was that the ability of either of two introns to stimulate expression declines as they are moved down a gene until all enhancement is lost about one kilobase from the start of the gene. This strongly suggests that introns boost expression by a co-transcriptional mechanism, possible by affecting transcript elongation. Because prior nuclear run-on transcription assays had failed to detect an effect of introns on transcription, the alternative method of chromatin immunoprecipitation was proposed as a means of detecting intron-mediated effects on the distribution of RNA polymerase II along a gene and to
detect biochemical modifications to the transcription machinery. Julie Pear was hired to perform this work but quit during the process of establishing the technique in my lab. Therefore, a one-year no cost extension was requested, and Shelley Martin was hired to continue this work. Shelley worked out many of the conditions needed to optimize the chromatin immunoprecipitation, but unfortunately has also resigned before this work was completed. A new technician is currently being sought, and potential collaborations are being explored so that these promising experiments can be conducted before the ending date of the funding period next July.
Impacts
The unparalleled biosynthetic capabilities of plants can be harnessed to synthesize many valuable substances such as medicines, edible vaccines, or unusual oils. In most cases, success depends on making large amounts of a specific protein, and ways to increase gene expression are enormously beneficial. Most of the candidate genes for such practical applications will be from non-plant sources and will lack introns naturally (e.g. viral or bacterial genes), or their introns will have been removed because plants are poor at splicing foreign introns. The expression of many of these genes in plants will be increased by the addition of a plant intron. Thus, a collection of easily inserted introns, and knowledge of how best to use them, will help boost the production of valuable proteins for scientific, agricultural, or commercial purposes. In addition to these practical considerations, the study of introns will continue to increase our understanding of the fundamental
events of gene expression in plants. Introns can significantly influence the expression of genes, and in some cases appear more important than the promoter in determining the specific tissues in which the gene is active.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
The goal of my research is to understand how introns enhance gene expression in plants. Most of the progress made this year has been towards the aim of identifying the intron sequences that are responsible for the very different enhancement mediated by the UBQ10 and COR15a introns. These introns are almost identical in length and composition but stimulate mRNA accumulation to very different degrees (15-fold vs. 2.5-fold.) The goal was to localize the sequences that control the magnitude of enhancement by creating a series of deletions in the UBQ10 intron and making hybrids between the UBQ10 and COR15a introns. Four deletions that together span the UBQ10 intron, and six hybrid introns, were made and put into plants. Even though quantitative measurements of expression will require the isolation of single-copy lines, a preliminary indication of the effect of each intron was obtained by histochemical staining. Tentative conclusions are that the sequences responsible for
enhancement are redundant and distributed throughout the UBQ10 intron, and that the COR15a intron does not contain a discrete sequence that inhibits enhancement. In a complementary approach, intron sequences that may be involved in enhancement are being sought by computational means in collaboration with Dr. Ian Korf at UC Davis. In essence, an algorithm (named the IMEter) was devised that measures the degree to which a test intron resembles the introns from highly conserved genes. Three lines of evidence suggest that the IMEter can predict the enhancing capability of introns. The first is that there is a correlation between the algorithm output and the carefully measured ability of introns to stimulate gene expression. The second is that virtually all introns that have been shown by other labs to enhance expression in Arabidopsis give high IMEter scores. The third is that a bulk analysis of all Arabidopsis introns revealed that first introns as a group give the highest IMEter scores,
which drop with intron number until sixth introns and then level off. This pattern is in excellent agreement with previous observations that the ability of an intron to boost expression declines as it is moved down a gene. Thus, the IMEter has uncovered an unexpected difference in intron structure based on position.
Impacts
The unparalleled biosynthetic capabilities of plants can be harnessed to synthesize many valuable substances such as medicines, edible vaccines, or unusual oils. In most cases, success depends on making large amounts of a specific protein, and ways to increase gene expression are enormously beneficial. Most of the candidate genes for such practical applications will be from non-plant sources and will lack introns naturally (e.g. viral or bacterial genes), or their introns will have been removed because plants are poor at splicing foreign introns. The expression of many of these genes in plants will be increased by the addition of a plant intron. Thus, a collection of easily inserted introns, and knowledge of how best to use them, will help boost the production of valuable proteins for scientific, agricultural, or commercial purposes. Identifying the intron sequences involved in enhancement will allow us to engineer a desired level of stimulation by changing the
sequence of an intron. The algorithm to predict the enhancing ability of introns will be particularly useful because it will allow new and stronger stimulatory introns to be found. The knowledge gained, and the algorithm to predict enhancement, can be adapted readily for use in many organisms including crop plants. In addition to these practical considerations, the study of introns will continue to increase our understanding of the fundamental events of gene expression in plants.
Publications
- Belostotsky, D.A. and A.B. Rose (2005). Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events. Trends in Plant Science. 10:347-353.
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Progress 01/01/04 to 12/31/04
Outputs
The goal of my research is to understand how introns enhance gene expression in plants. The two aims of this project are to investigate the effects of varying intron position within a gene, and to explore the effect of introns on transcription using chromatin immunoprecipitations. The first of these aims has been completed and the results were recently published. To test the importance of intron location, two series of reporter genes were constructed. Each series consists of eight fusions between the Arabidopsis PAT1 gene and the GUS reporter gene that differ only in the position of the single intron they contain. In six, the intron is in protein coding sequences, while the other two have an intron in the 3'-UTR. The series differ in the introns used, either PAT1 intron 1 or UBQ10 intron 1, which stimulates mRNA accumulation to a greater degree. Several single-copy homozygous transgenic Arabidopsis lines were identified for each of the sixteen constructs, and splicing
efficiency, steady-state GUS mRNA levels, and enzyme activity was measured in all. For both series of fusions, the amount of mRNA that accumulated declined as the intron was moved down the gene until all stimulatory effects were lost when the intron was about a kilobase from the start of transcription. Even though the two introns tested boost expression by different amounts, they had similar positional requirements. Both introns also stimulated GUS enzyme activity about twice as much as they increased GUS mRNA levels relative to the intronless control, regardless of intron location. The stimulation mediated by two of four other introns tested at the position nearest the promoter also was greater at the level of GUS activity than mRNA accumulation. These findings suggest that introns affect gene expression by at least two distinct mechanisms. One is an intron-specific and position dependent effect on transcription, while the other is a smaller effect on translational efficiency that is
less influenced by intron identity or location. Progress has been made on the second aim, the use of chromatin immunoprecipitation to explore the effect of introns on transcription. The chromatin isolation and sonication procedures have been established, the anti-polymerase antibody has been shown to recognize the Arabidopsis protein in western blots, and the primers and conditions for PCR amplification of different parts of the GUS reporter have been worked out. Our attention is currently focused on increasing the yield from the immunoprecipitations.
Impacts
The biosynthetic abilities of plants are beginning to be harnessed to produce many valuable substances such as medicines, biodegradable plastics, or unusual oils. Most of these practical applications depend on the expression of genes from other organisms, and success is usually determined by the amount of gene product made. Because introns enhance gene expression in plants, and most of the candidate genes lack introns naturally (bacterial and viral genes) or have had their introns removed because plants are poor at splicing foreign introns, the method I devised to easily insert plant introns into any gene has potential to be enormously beneficial. Such tools are valuable to the extent that we know how to use them. Thus, the basic knowledge gained from these studies will inform choices about which intron to use and where to place it for maximum effect. Furthermore, these findings have revealed basic features of gene expression in plants, an important area whose study
has been hampered by lack of in vitro assays. My work has allowed a comparison of gene expression in plants with that in other organisms, highlighting some important similarities and differences.
Publications
- Rose, A.B. (2004). The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. The Plant Journal 40:744-751.
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Progress 01/01/03 to 12/31/03
Outputs
The goal of my research is to understand how introns enhance gene expression in plants. The hypothesis that introns elevate mRNA accumulation primarily by increasing transcript elongation will be tested by varying the position of an intron in a gene, and by measuring the distribution and modification of RNA polymerase II on genes containing or lacking an intron using chromatin immunoprecipitations. In the five months since the start of funding, significant progress has been made on the first of these aims. Two series of reporter genes were previously constructed. Each series consists of eight fusions between the Arabidopsis PAT1 gene and the GUS reporter gene that differ only in the location of the single intron they contain. In six, the intron is in protein coding sequences, while the other two have an intron in the 3'-UTR. The series differ in the introns used, either PAT1 intron 1 or UBQ10 intron 1, which stimulates mRNA accumulation to a greater degree. Several
single-copy homozygous transgenic Arabidopsis lines have now been identified for each of the sixteen constructs, and preliminary expression data has been obtained for all. The ability of both introns to enhance expression declines with distance from the promoter. The decline appears more gradual for the PAT1 intron than the UBQ10 intron, but neither intron has a significant effect when more than one kb from the start of transcription. The differences in GUS activity derived from the different constructs is mostly due to differences in mRNA accumulation, although there may also be a slight effect of introns on translation. The exon junction complex deposited on mRNA during splicing may stimulate translation in plants as it does in mammalian cells. The simplest explanation for position-dependent enhancement is that transcription of an intron near the start of a gene causes a change in the polymerase that significantly increases the amount of mature mRNA produced. Different introns could
mediate this change to different degrees, but the polymerase remains responsive to stimulation for only the first kilobase of transcription. After that, full-length transcripts can still be produced at a basal rate but introns provide no enhancement.
Impacts
Plants can be engineered to produce many valuable substances, such as medicines, biodegradable plastics, or unusual oils. This study of introns and their effect on gene expression will provide practical knowledge and useful tools to help maximize yields, and will increase our understanding of the basic mechanisms that control the amount of protein produced from a gene.
Publications
- No publications reported this period
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