SUMMARY DNA glycosylases are critical enzymes that recognize small base lesions in DNA and trigger their repair to preserve genome integrity. The Arabidopsis MBD4‐like (MBD4L) DNA glycosylase improves tolerance to genotoxic stress. This enzyme is encoded by a single gene carrying an exonic intron (exitron) at its 5′ region. Although alternative splicing (AS) of exitrons (protein‐coding cryptic introns within exons) is suspected to increase protein diversity, phenotypes associated to exitron removal or retention are only known for a few genes. Here, we show that AS of the MBD4L exitron determines the generation of two enzyme isoforms with different subnuclear localization. Both isoforms conserve the catalytic domain but are directed to either the nucleoplasm or the nucleolus. Interestingly, heat stress regulates the AS of the MBD4L exitron and increases the relative abundance of the nucleolar variant. This process depends on the splicing factors NTR1 and RS31. Next, we generated transgenic plants expressing a mutated MBD4L‐GFP gene version that abolished exitron splicing and found that nucleolar protein targeting was impaired in these plants. Our findings suggest that AS of the MBD4L exitron can function as a mechanism to drive the enzyme to the nucleolus during heat stress. Several DNA repair enzymes reach the nucleolus under particular conditions although AS of exitrons has not been so far associated with this process. To our knowledge, this is the first example of an exitron mediating enzyme localization in eukaryotes.
SUMMARYDNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl‐CpG‐binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4‐like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5‐fluorouracil (5‐FU) and 5‐bromouracil (5‐BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5‐FU and 5‐BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER‐associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5‐BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5‐FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG‐GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L‐deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.
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