Progress 08/01/06 to 07/21/09
Outputs
OUTPUTS: The main activity during the funding period was to conduct and analyze all of the experiments proposed. This included the training of numerous students and postdoctoral scientists. In the Rose lab, a total of six undergraduate students were involved in performing the research, as were three graduate students doing laboratory rotations. In addition, two technicians were hired for a combined total of sixteen months. The Korf lab trained two postdocs and two graduate students in the computational analysis of introns. The services provided during the period of funding included consulting for a locally owned small biotechnology company that is interested in using introns to increase gene expression in insect cells. A tangible product to arise from this research is the IMEter, which is software that reliably predicts the enhancing ability of introns in rice and Arabidopsis. An online version of the IMEter is available for public use at http://korflab.ucdavis.edu/cgi-bin/web-imeter.pl and a link to the IMEter was established in the TAIR database as a Gene Expression Resources portal (http://www.arabidopsis.org/portals/expression/index.jsp). In addition to the publications listed below, the results of this research were disseminated through the presentation of posters at four scientific meetings (the Cold Spring Harbor Eukaryotic mRNA Processing meetings in August 2007 and 2009, the Biology of Post-transcriptional Gene Regulation Gordon Conference in July 2008, and the Plant Gene Expression Center 20 Year Symposium in Berkeley, June 2007). Also, Dr. Korf gave a talk on this subject at the Biology and Mathematics in the Bay Area meeting in November 2008. PARTICIPANTS: Alan B. Rose, PI, carried out the expression experiments, constructed plasmids and did plant transformations, and supervised the students in his lab. This included six undergraduate students (Aruna Dutta, Tamika Rozema, Janice Kwong, Angie Hood, Ilona Gurkova, and Jeff Lu) who mostly performed plasmid construction and helped with the growth and maintenance of the plants. Three graduate students (Amanda Schrager, Daniel Chu, and Phillip Conklin) performed laboratory rotations in the Rose lab, during which they constructed plasmids, did preliminary expression studies, or isolated introns from rice and Arabidopsis. Two people worked in the Rose lab as Senior Research Associate II (Dinah Arumainayagam from Aug. 2007 to May 2008 and Shelley Martin from Aug. 2006 to Dec. 2006), doing plasmid construction and helping with expression experiments. The Co-PI, Ian F. Korf, performed most of the initial software development and supervised the people in his lab. This included two graduate students (Shahram Emami and Tali Elfersi) and two postdocs (Genis Parra and Keith Bradnam), all of whom helped to test and refine the IMEter algorithm. TARGET AUDIENCES: The target audiences for this work include scientists involved in basic research as well as those involved in biotechnology applications in which a high level of gene expression is desired. The work provided insights into the general mechanisms of eukaryotic gene expression and is expected to be of interest as a fundamental biological process. The success of many biotechnology ventures often rests on the ability to produce a large amount of product from a specific gene, and scientists attempting this would benefit from the increased yield that introns can provide. Intron-mediated enhancement has been observed in many diverse organisms in addition to plants, making this research relevant to a broad audience. PROJECT MODIFICATIONS: None. The project was completed as proposed.
Impacts
The revised objectives for this funding period were to identify the intron sequences responsible for the very different enhancement mediated by the UBQ10 and COR15a introns, and to test, refine, and adapt for crop plants the IMEter algorithm. Both of these aims were fully accomplished, and the results generated new insights into intron structure and the ways introns could be stimulating gene expression. One change in knowledge to result from this work is a new appreciation that introns early in genes are compositionally distinct form those that occur further from the start of transcription, and that this difference in composition is related to the effect of promoter-proximal introns on gene expression. Another is that the intron sequences responsible for boosting gene expression are distributed throughout enhancing introns. This is significant because it eliminates all possible mechanisms that involve the binding of a factor to a specific or localized sequence, or that depend on a particular RNA secondary structure. Prior to this work, the only way to identify introns that boost gene expression for scientific purposes or for plant biotechnology was trial and error. The development of the IMEter algorithm greatly facilitates the identification of enhancing introns, a change of action that should be of significant benefit for all practical applications in which a high level of gene expression is desired. In the future, the ability to increase expression of a specific gene through the use of stimulating introns has potential to change conditions by improving the nutritional quality of foods and by providing a means to generate new value-added crops.
Publications
- Rose, A.B. (2007) Book Review: Plant Gene Expression. Science STKE, pe26.
- Korf, I.F. and A.B. Rose (2009) Applying word-based algorithms: The IMEter. In Plant Systems Biology (D. Belostotsky ed.) Methods in Molecular Biology (Clifton, NJ) 553:287-301.
- Rose, A.B., T. Elfersi, G. Parra, and I. Korf (2008) Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell 20:543-551.
- Rose, A.B. (2008). Intron-mediated regulation of gene expression. In Nuclear pre-mRNA Processing in Plants (A.S.N. Reddy and M. Golovkin, eds.) Current Topics in Microbiology and Immunology 326:277-290.
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: The goal of my research is to understand how introns enhance gene expression in plants. The aims of the current funding are to localize the intron sequences responsible for boosting expression using deletions and hybrids between two introns that affect expression to different degrees, and to test, refine and expand an algorithm for predicting the enhancing ability of introns. Significant progress has been made on both of these aims this year. Measurements of the mRNA accumulation and enzyme activity derived from reporter genes containing hybrid or deletion-containing introns confirm that the sequences responsible for stimulating expression are distributed throughout the enhancing UBQ10 intron. That is, the removal of any part of this strongly enhancing intron has a negligible effect on its ability to boost expression, and all regions of this intron have some enhancing capacity when inserted into a non-stimulating intron. The dispersed nature of the enhancing sequences indicates that introns must influence gene expression by a unique mechanism. The algorithm used in the second aim is a word-based method for comparing an individual intron to promoter-proximal introns as a group. We had previously noted a strong correlation between the ability of an intron to stimulate expression and the score it generates with this algorithm. Six new introns were tested and all increased expression in proportion to their score, demonstrating that the algorithm is capable of predicting the enhancing ability of introns. The quantitative data from these six introns and the six previously tested in the same gene were used to optimize the variable parameters of the algorithm. The algorithm was adapted to use rice intron sequences, and this version gives a high score to virtually all introns known to increase expression in rice and most that boost expression in maize and Arabidopsis. Motifs were identified that are over-represented in the highest scoring introns, and also by using the algorithm to scan intron sequences in a small sliding window. Both methods suggest the sequence TCGATT is important in generating high scores and therefore may be involved in the mechanism of enhancement. The algorithm was also incorporated into a computational approach to design synthetic introns with higher scores than any natural intron. These could be useful if they stimulate more strongly than existing introns, and it provides a way to generate an infinite supply of new enhancing introns in a variety of species. The algorithm is now publicly available from a web site (http://korflab.ucdavis.edu/cgi-bin/web-imeter.pl), and a link to this web site has been established in the TAIR database of Arabidopsis genome information as a Gene Expression Resources portal. (http://www.arabidopsis.org/portals/expression/index.jsp). PARTICIPANTS: Alan Rose performed and supervised all of the research involving plants and plasmid construction. Ian Korf performed and supervised all of the computational research. During the period covered by this report, this research provided training for two graduate students doing laboratory rotations and three undergraduates doing research for course credit. Research collaborations were recently initiated with two other groups (Karen McDonald, in Chemical Engineering at UC Davis) and Tracy Johnson (UC San Diego). TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The benefits of this research will be both practical and scientific. The success of many biotechnology ventures depends on the production of large amounts of a specific protein. Most of the candidate genes involved in such applications will be from other species and will lack introns naturally (e.g. viral or bacterial genes), or their introns will have been removed to avoid problems of splicing foreign introns. The expression of many of these genes will be increased by the addition of an intron from the host species. 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. 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 or made. Identifying the intron sequences involved in enhancement will allow us to engineer a desired level of stimulation by changing the sequence of an intron, and provides the basis for future biochemical isolation of other factors involved in the mechanism of enhancement. 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 and other organisms. An indication of the significance of this work is that the article reporting the results was featured in the In Brief section of the Plant Cell (Farquharson, K.L. 2008. The IMEter Predicts an Intron's Ability to Boost Gene Expression. Plant Cell 20:498), and has been evaluated by Faculty of 1000 Biology (http://www.f1000biology.com/article/id/1104324/evaluation).
Publications
- Rose, A.B. 2007. Book Review: Plant Gene Expression. Science STKE, pe26.
- Rose, A.B., Elfersi, T., Parra, G., and Korf, I. 2008. Promoter-Proximal Introns in Arabidopsis thaliana are Enriched in Dispersed Signals that Elevate Gene Expression. Plant Cell 20: 543-551.
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Progress 08/01/06 to 08/01/07
Outputs
The goal of my research is to understand how introns enhance gene expression in plants. The aims of the current funding are to analyze hybrid introns formed using parts of two different introns, only one of which significantly boosted gene expression, and to test, refine and expand an algorithm for predicting the enhancing ability of introns. Since funding for this project began in August 2006, significant progress has been made on both of these aims. For the first aim, homozygous single-copy transgenic lines containing a reporter gene with one of twelve different hybrid introns or controls were identified. For each, enzyme activity, mRNA accumulation, and intron splicing efficiency have been determined, with the following findings. First, the minor changes made to introduce restriction sites to facilitate the construction of hybrid introns have no discernible effect on the enhancing effect of the two parental introns. Second, that the enhancing sequences are roughly
evenly distributed throughout the first three quarters of the enhancing intron, and that each enhancing region has an additive effect on expression. Third, any hybrid intron containing enhancing sequences at the beginning of the intron seem to have an additional two-fold effect on translational efficiency. That is, the enhancement seen at the level of enzyme activity is twice that seen at the level of mRNA accumulation, as if more enzyme is being produced per unit of mRNA. Perhaps the splicing of those introns promotes either export of the mRNA from the nucleus to the cytoplasm or association of the mRNA with ribosomes. Fourth, that all of the hybrid introns are completely spliced, indicating that any differences between them are not due to splicing efficiency. To test the predictive abilities of the algorithm (the IMEter) in the second aim, transgenic lines with a single copy of a reporter gene containing introns predicted to differ in their effect on expression have been identified.
Of the eight introns, two clearly prevent expression of the reporter. The annotation for one of these was recently modified in the Arabidopsis database to indicate that the intron is actually 38 nt shorter than originally predicted, which would cause a reading frame shift. Expression studies on the remaining 6 introns will begin in the new year. An analysis of introns in rice, Drosophila, and C. elegans using the IMEter has revealed than first introns are structurally different than later introns in Arabidopsis, rice, and Drosophila, but not in C. elegans. A program has been devised to very rapidly evaluate the effect of changing various parameters of the IMEter and to incorporate new information as it is generated, with the goal of optimizing the correlation between IMEter scores and expression data. The IMEter is currently being used on the hybrid introns in hopes of pinpointing sequences that are potentially responsible for the enhancement observed.
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
- No publications reported this period
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