Alternative splicing of an exitron determines the subnuclear localization of the Arabidopsis DNA glycosylase MBD4L under heat stress

Abstract

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.

Alternative splicing as a key player in the fine‐tuning of the immunity response in Arabidopsis

“…Although generation of functional proteins by alternatively spliced exitrons is still poorly documented, there are examples of exitron‐spliced protein isoforms involved in stress response. These include ABI3 variants, which regulate seed abscisic acid sensitivity (Punzo et al, 2020b), FLS2‐derived suppressors of signalling mediated by the pathogen effector flagellin (flg22) (Cheng et al, 2020), or MBD4L isoforms generated during heat stress that have different subnuclear localization (Cecchini et al, 2022). All these observations support AS‐mediated stress adaptation of plant transcriptomes and proteomes.…”

Regulatory Network of Serine/Arginine-Rich (SR) Proteins: The Molecular Mechanism and Physiological Function in Plants

“…Besides the effect on the growth and development, the SR proteins work in response to stresses and hormones [ 81 , 82 , 83 , 84 , 85 , 86 ]. The hypersensitivity of expression regulation in response to environmental stresses of SR genes has been well studied in Arabidopsis and rice [ 66 , 81 ].…”

Noncatalytic Domains in DNA Glycosylases

“…Interestingly, plant homologs of MBD4 lack an MBD but retain the long N-terminal extension [163]. Alternative splicing in this region produces protein isoforms with different intranuclear localization and different redistribution response to heat stress [164].…”