|NAYAK, NIHAR - University Of Kentucky|
|PUTNAM, ANDREA - Case Western Reserve University (CWRU)|
|ADDEPALLI, BALASUBRAHMANYA - University Of Cincinnati|
|LOWENSON, JONATHAN - University Of California|
|CHEN, TINGSU - University Of Kentucky|
|JANKOWSKY, ECKHARD - Case Western Reserve University (CWRU)|
|PERRY, SHARYN - University Of Kentucky|
|LIMBACH, PATRICK - University Of Cincinnati|
|CLARKE, STEVEN - University Of California|
|DOWNIE, BRUCE - University Of Kentucky|
Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2013
Publication Date: 7/31/2013
Citation: Nayak, N., Putnam, A., Addepalli, B.M., Lowenson, J., Chen, T., Jankowsky, E., Perry, S.E., Dinkins, R.D., Limbach, P.A., Clarke, S.G., Downie, B. 2013. An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase. The Plant Cell. 25:2573-2586.
Interpretive Summary: Orthodox seeds are capable of withstanding dehydration to ~5% moisture, then entering a period of quiescence and maintain viability for months, or years in some species. This capacity is the basis of agriculture, allowing a portion of harvested seed to be retained to produce next season’s crop. During this dehydrated state, proteins can undergo spontaneous degradation to form non-functional proteins that either need to be replenished, or repaired, for viability. For de novo synthesis, there is a requirement for transcription and translation. It has been hypothesized that the proteins part of protein synthesis, or the translational machinery, may be a major target requiring protection and repair for seed viability. The Protein Isoaspartyl Methyltransferase (PIMT) protein has been shown to play a role in seed viability, as longevity is compromised mutants deficient in PIMT. The PIMT protein is involved in protein repair, but the targets of PIMT have not been characterized. In this work we demonstrate that one putative target of the Arabidopsis thaliana PIMT1 protein is the A. thaliana Plant RNA Helicase75 (AtPRH75) protein, a presumptive RNA helicase that is predicted to be involved in translation and/or in ribosome maturation. We found that severe mutations in the AtPRH75 gene are lethal during embryogenesis indicating that loss of PRH75 enzymatic activity can have dire consequences for the plant. We further demonstrate that the AtPRH75 protein can be rapidly compromised by heat treatment, and that inclusion of PIMT in vitro can rescue the PRH75 activity, suggesting that PRH75 is one of the targets of PIMT.abolishes activity of an RNA helicase whose function is required for normal seed development.
Technical Abstract: Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by thermal insult (37°C) but unwinding activity was considerably recovered following incubation with PIMT in the presence of AdoMet. However, recovery was severely curtailed with prolonged (8h and longer) prior exposure to 37°C. A distinct alteration was discernible in circular dichroism spectra from active, compared to inactive (4- or 8-h heat-treated), PRH75. Trypsin digests of PRH75 exposed to 37 °C followed by LC-MS/MS identified isoaspartyl residues in the DEAD box motif arising at 4- (D258) and 12-h (D255) of thermal insult, both repairable by PIMT. The decline in the ability of PIMT to recover PRH75 RNA unwinding activity over time may be due to accumulated, morphological alteration of the PRH75 protein. Parameters defining Boltzmann equation fits describing thermal denaturing curves of PRH75, before or after prior exposure to 37°C, indicated that PRH75 heat stability was progressively compromised, correlating with the time-dependent loss of PIMT-repair capacity. To examine the physiological consequences of a loss of PRH75 RNA unwinding activity in vivo, the embryo lethal phenotype of plants deficient in PRH75 were examined. Complementation of a prh75 mutant with 35Spro:PRH75 obviated lethality. Persistent screening of the least deleterious (based on large variations in the stage of embryo arrest and the position of the T-DNA insertion) mutant in the allelic series resulted in the recovery of a prh75 mutant. These plants had reduced fertility and could produce misshapen seeds. These results demonstrate that isoAsp formation in PRH75 abolishes activity of an RNA helicase whose function is required for normal seed development.