Abstract
Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 ( sor1). The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth.
Highlights
The 26S proteasome (26SP) is a multisubunit protease responsible for the degradation of proteins that are covalently labeled with a polyubiquitin (Ub) chain via the combined action of Ub activating enzymes, Ub conjugating enzymes and Ub ligases[1]
These lines were generated by transforming Arabidopsis plants (C24 accession) with a T‐DNA construct that contains a pro‐ moterless neomycin phosphotransferase gene (NPTII) without a starting methionine which is preceded by a 3 ́ splice site of the first intron of the apurinic endonuclease (APR)[14]
We have previously shown that this predicted latent 5 ́ splice site is STOP codon‐associated, and that the pre‐mRNA splicing of the chimeric intron leads to the produc‐ tion of the fusion mRNA15
Summary
The 26S proteasome (26SP) is a multisubunit protease responsible for the degradation of proteins that are covalently labeled with a polyubiquitin (Ub) chain via the combined action of Ub activating enzymes, Ub conjugating enzymes and Ub ligases[1]. The rpn12a‐1 mutation is unusual because the T‐DNA is inserted downstream of the RPN12a gene, and both the full‐length RPN12a cDNA and a chimeric RPN12a‐NPTII cDNA are produced[15] This suggested that two types of cis signals involved in the pre‐mRNA processing of RPN12a are competing. As a result of the action of these two op‐ posing pre‐mRNA processing mechanisms, one part of the mRNA species transcribed from the mutant RPN12a gene is translated into a functional RPN12a protein, and the other is translated into a chi‐ meric RPN12a‐NPTII fusion protein Because both RPN12a forms are incorporated into the 26SP, the total proteasome activity in these mutant seedlings is reduced, but not abolished[15]. This finding reinforces our previous conclusion that proteasome function is critical for the cytokinin-dependent regulation of plant growth
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
