Minisymposium 19: Gene regulation

Abs # 34003: Maintenance of a functional proteome.

Presenter:	Downie, Bruce , adownie@uky.edu
Authors	Xu, Qilong  (A)   Villa, Sarah  T. (B)   Belcastro, Marisa  P. (A)   Dinkins, Randy D. (A)   Clarke, Steven . G (B)   Downie, Bruce  (A)
Affiliations:	(A): University of Kentucky (B): University of California Los Angeles

The non-enzymatic conversion of aspartate and asparagine residues to the abnormal isoaspartate is thought to be a predominate form of deleterious, covalent protein modification in cells. Fortunately, the repair enzyme, Protein L-isoaspartyl methyltransferases (PIMT), can iteratively methylate abnormal isoaspartyl residues leading to conversion to L-aspartate. The model plant Arabidopsis thaliana is unique among eukaryotes studied to date in that it possesses two genes encoding PIMT, PIMT1 (At3g48330) and PIMT2 (At5g50240). The second PIMT exhibits a complex transcriptional control involving different transcriptional initiation sites (TIS) and 5'- and 3'-alternative splice site selection in the first intron. Transient expression of gene fusions between each of the PIMT2 cDNAs from one of the three TIS (I, II, and III) and GFP in onion epidermal peels or tobacco leaf discs indicated that TIS I-PIMT2 was sequestered in the nucleus, TIS II-PIMT2 is targeted to the mitochondria and plastids, while TIS III-PIMT2 is cytoplasmically localized. Alteration of the canonical nuclear localization signal abolished the strict nuclear residency resulting in TIS I-PIMT2:GFP throughout the cell. Recombinant proteins generated from cDNAs from each TIS encoded protein with an L-isoaspartyl methyltransferase activity. Multiplex RT-PCR was used to establish PIMT1 and PIMT2 transcript presence and abundance, relative to β-TUBULIN, in various tissues and under a variety of stresses imposed on seeds and seedlings. PIMT1 transcript is constitutively present but can increase, along with PIMT2, in developing seeds presumably in response to increasing endogenous ABA. Transcript from PIMT2 also increases in establishing seedlings due to exogenous ABA or applied stress presumably through an ABA-dependent pathway. Furthermore, Cleaved Amplified Polymorphic Sequence analysis and nondenaturing PAGE of PIMT2 amplicons revealed that the ratio among the 3' splice site selection variants alters upon ABA application. Leaves from seedlings growing on MS media synthesized PIMT2 3' splicing variants in approximately equal abundance but those from seedlings on MS media supplemented with 100µM ABA had a predominance of the shorter splicing variant, implicating a role for the spliceosome in orchestrating the plant's response to stress. Of the four possible PIMT2 splicing variants resulting from alternative 5' and 3' splice site selection, only 3 have been confirmed to occur in vivo.