CPR5 modulates plant immunity via RNA processing

Immune systems are double-edged swords. We need them to stay healthy, but mis-regulation of immunity genes can cause autoimmunity, often leading to dwarfism and cell death. There are dozens of autoimmune diseases in humans, including Type I diabetes, rheumatoid arthritis, multiple sclerosis, and lupus. Plants have evolved sophisticated systems to regulate the expression of immunity-related genes and avoid autoimmune responses such as spontaneous lesion formation and growth inhibition (see Chakraborty et al., 2018; Ngou et al., 2022). In this issue, Shun Peng and colleagues (Peng et al., 2022) describe a new layer of this regulatory system with the discovery that Arabidopsis thaliana CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES 5 (CPR5) regulates transcript levels of immunity-related genes by modulating both alternative splicing and polyadenylation. The Arabidopsis cpr5 mutant was isolated and described 25 years ago in a screen for constitutive expression of defense marker genes (Bowling et al., 1997). The CPR5 protein was later found to be a nucleoporin that influences plant immunity via a role in nuclear transportation and regulation of the cell cycle (Wang et al., 2014; Gu et al., 2016), but its precise function has remained elusive.

Peng et al. started with a forward genetic screen for suppressors of the cpr5 mutant and identified two factors, PLEIOTROPIC REGULATOR 1 (PRL1) and FACTOR INTERACTING WITH POLY(A) POLYMERASE 1 (FIP1), which were synergistically required for the cpr5 mutant phenotype. PRL1 encodes a pre-mRNA splicing factor that is part of the NineTeen Complex (NTC) that activates the spliceosome. FIP1 is a component of the CLEAVAGE AND POLYADENYLATION SPLICING FACTOR (CPSF), which is part of the multiprotein complex that regulates pre-mRNA polyadenylation. Moreover, they discovered that CPR5, PRL1, and FIP1 physically interact in planta on the nuclear envelope and in nuclear speckles. Nuclear speckles are enriched in splicing factors and serve as a site for a number of RNA processing steps (see Figure).

Figure.

Localization and functional aspects of CPR5. Bimolecular complementation assay demonstrates interaction of CPR5 with PRL1 and FIP1 in the nuclear envelope and nuclear speckles (top panel). RIP analysis shows that CPR5 binds AGO1 (bottom left) and PCR results show the splicing pattern of AGO1 transcripts (bottom right). NS, nuclear speckles. Adapted from Peng et al. (2022), Figure 4–6.

To further explore the role of CPR5 in pre-mRNA processing, the authors performed RNA-seq analysis on wild-type, cpr5 single mutant, prl1 fip1double mutant, and cpr5 prl1 fip1 triple mutant plants to identify changes in alternative splicing in these mutants. They also performed polyadenylated site sequencing (PAS-seq) on these lines to identify changes in polyadenylation. These analyses revealed that two-thirds of the differentially expressed genes in the cpr5 mutant plants were dependent on PRL1/FIP1, including important plant immune response marker genes. However, the overlap between cpr5-upregulated genes, alternatively spliced genes, and alternatively polyadenylated genes was limited, indicating that the CPR5-NTC/CPSF complex affects a different set of genes in terms of RNA transcription, splicing, and polyadenylation. Thus, these results overall pointed to a pivotal role for CPR5 in RNA processing, particularly in pre-mRNA splicing.

Next, the authors focused on ARGONAUTE1 (AGO1), an alternatively spliced gene that functions in gene silencing and in the regulation of gene expression in the nucleus (Bajczyk et al., 2019). They found that the cpr5 phenotypes of constitutive immune responses and early senescence are suppressed by ago1, suggesting that AGO1 functions downstream of CPR5 to activate plant-programmed cell death and immunity. RNA binding assays demonstrated that the RNA recognition motif (RRM)-domain of CPR5 can bind RNA, including the transcripts of AGO1, which may lead to its alternative splicing (see Figure).

In sum, the authors provide convincing evidence that links plant immunity with RNA-processing. CPR5 fulfills an interesting role linking nuclear transportation, cell cycle progression, and RNA processing. More work is needed to investigate how CPR5 functions, how it is translocated, and how it affects the translocation of other proteins between the nuclear envelope and the nuclear speckles.