Progress 09/01/02 to 08/31/04
Outputs
Arabidopsis possesses four genes that encode poly(A) polymerase (PAP). These genes are situated on chromosomes I, II, III, and IV, respectively (and are termed in this report according to their respective chromosome). Based on the results of RNAi studies and/or T-DNA insertion mutant analysis, we can conclude that all four genes are essential for some aspect of Arabidopsis growth and development. These results are of interest as they indicate that the four PAP genes have non-redundant roles in plant growth and development. Whether this is due to differing expression characteristics, or different properties of the respective enzymes, is under study at this time. Analysis of promoter-GUS fusions for the four PAP promoters has been completed.. The PAP(I), PAP(II), and PAP(IV) promoters are found to be most active in reproductive structures. However, there are mutually-exclusive aspects to the activity profiles, an observation that may help explain the essentiality of the
PAP(II) and PAP(IV) genes. The PAP(III) promoter is most active in root tips and leaf primordial, as well as male-specific reproductive tissues. The protein products of the PAP(I), PAP(II), and PAP(IV) genes all localize to the cell nucleus, based on the distribution of PAP-GFP fusion proteins in transiently-transfected cells. Interestingly, however, the PAP(III) protein does not accumulate in the nucleus, but rather is distributed in a non-random manner in the cytoplasm. This suggests that the product of the essential PAP(III) gene is cytoplasmic, and raises important new questions regarding the polyadenylation of mRNAs in the cytoplasm of plant cells.
Impacts
Defining the role(s) that the different Arabidopsis poly(A) polymerases play will further our understanding of gene expression in plants. This insight will contribute to a improved capabilities vis-a-vis the development of improved technologies for transgene expression, and to a greater ability to conceptually integrate this aspect of gene expression with other steps in the process.
Publications
- Addepalli, B., Meeks, L. R., Forbes, K. P., and Hunt, A. G. (2004) Novel alternative splicing of mRNAs encoding poly(A) polymerases in Arabidopsis. Biochimica Biophysica Acta 1679, 117-128.
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Progress 09/01/02 to 08/31/03
Outputs
Based on the results of RNAi studies and/or T-DNA insertion mutant analysis, we can conclude that PAP(II), PAP(III), and PAP(IV) are all essential for some aspect of Arabidopsis growth and development. Results have not yet been obtained for PAP(I), but these should be available within the next 3 months. With the completion of the studies on PAP(I), this specific aim will have been completed. These results are of interest as they indicate that the four PAP genes have non-redundant roles in plant growth and development. Whether this is due to differing expression characteristics, or different properties of the respective enzymes, is under study at this time. Northern blot analysis indicated that the PAP(I), PAP(II) and PAP(IV) genes were most highly expressed in reproductive tissues, and the PAP(II gene was the predominant gene expressed in leaf tissues. Analysis of promoter-GUS fusions corroborates these findings. In particular, the PAP(I), PAP(II), and PAP(IV)
promoters are found to be most active in reproductive structures. However, there are mutually-exclusive aspects to the activity profiles, an observation that may help explain the essentiality of the PAP(II) and PAP(IV) genes. Fusions involving the PAP(III) gene are presently being analyzed. A two-hybrid screen for interactions between PAP(III) or PAP(IV) and all other polyadenylation factor subunits whose clones are presently available has been completed; this screen indicates that Arabidopsis PAPs interact with four other Arabidopsis polyadenylation factor subunits: CPSF160, CPSF100, CPSF30, and Fip1p. No interactions were observed with the other homologues (six) for which clones are available. Equivocal results were obtained with one subunit (CstF50), and these are being confirmed. No differences were observed when assays were done with PAP(III) or PAP(IV) (the latter is representative of the other two PAPs, while the former is novel in size and sequence). This screen reveals a
number of interactions that are also seen in other systems, but at least one (between PAP and CPSF100) that has only been reported in Arabidopsis. The absence of a PAP-CFIm-25 interaction also distinguishes plants from mammals. The PAP-CPSF100 interaction has been confirmed by in vitro pull down assayss. In preliminary studies, the Arabidopsis Fip1p homologue affects PAP much as does its yeast counterpart - PAP activity is modestly increased, but the processivity of the enzyme is dramatically reduced.
Impacts
Defining the role(s) that the different Arabidopsis poly(A) polymerases play will further our understanding of gene expression in plants. This insight will contribute to a improved capabilities vis-a-vis the development of improved technologies for transgene expression, and to a greater ability to conceptually integrate this aspect of gene expression with other steps in the process.
Publications
- Elliott, B. J., Dattaroy, T., Meeks, L. R., Forbes, K. P., and Hunt, A. G. (2003). An interaction between an Arabidopsis poly(A) polymerase and a homologue of the 100 kD subunit of CPSF. Plant Molecular Biology 51, 373-384.
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Progress 09/01/02 to 12/31/02
Outputs
This project was initiated on Sept. 1, 2002. In the first four months of the project, expression constructs for antisense and RNAi ablation of expression of the four Arabidopsis poly(A) polymerase genes were completed, and transformations begun.
Impacts
It is expected that a better understanding of the roles of each of the four Arabidopsis poly(A) polymerases in gene expression will lead to improved insight into gene expression in plants. Additionally, novel means by which regulated control of the expression of foreign genes in plants may be developed.
Publications
- No publications reported this period
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