Role of Arabidopsis Pumilio RNA binding protein 5 in virus infection

Abstract

Regulation of gene expression is mediated by diverse RNA binding proteins which play important roles in development and defense processes. Pumilio/FBF (Puf) protein in mammals functions as a posttranscriptional/translational repressor by binding to the 3′ UTR regions of its target mRNAs. Previous study reported that APUM5 provides protection against CMV infection by directly binding to CMV RNAs in Arabidopsis. CMV RNAs contain putative Pumilio-binding motifs and APUM5 bound to the 3′ UTR and some of its internal motifs both in vitro and in vivo. APUM5 works as a negative regulator of the 3′ UTR of CMV and it might regulate CMV replication. Our findings suggest that APUM5 acts as a defensive repressor in plants during CMV infection. However, functions of APUM5 and other APUM members are still not clear and more studies are needed to find out the interacting partners and target mRNAs in host plant.

Pumilio/FBF (Puf) proteins have an evolutionary conserved Pumilio homology domain (PHD) and are found in organisms as diverse as protozoa, fungi, mammals and plants.^1-3 Puf is proposed to function as a posttranscriptional/translational repressor by interacting with sequence-specific motifs in the 3′ UTR of its target mRNAs⁴ or by associating with rRNA processing, possibly in a sequence non-specific manner in the nucleolus.^5,6 The targets of Puf proteins are involved in differentiation, development, cell cycle, stem cell renewal and synaptic functions.^7-9 In plant, Puf-like proteins are encoded by more copies of genes than mammalian Puf proteins. For example, Arabidopsis and rice genomes contain 25 and 19 putative Puf genes whereas human, Drosophila, C. elegans and yeast genomes contain two, one, nine and six Puf genes, respectively. In a bioinformatics analysis, the expression of some plant Puf genes increased in response to biotic and abiotic stresses and exhibited tissue-dependent differential expression.¹⁰This demonstrates that plant Puf proteins could play diverse roles in plant development, differentiation and biotic and abiotic stresses.

Recently, targets of the Arabidopsis Puf protein were selected by yeast three-hybrid (Y3H) screening using APUM2 as bait, and five target mRNA sequences were found.¹¹Unexpectedly, these candidate genes were not associated with well-known target genes, such as those involved in development, stem cell renewal and differentiation. One of the candidate target mRNAs was RD19 (At4g39090), RESPONSIVE TO DEHYDRATION 19, which responds to desiccation as an abiotic stress.¹¹ RD19 interacts with the Ralstonia solanacearum type III effector PopP2.¹² In terms of biotic stress regulation, the direct binding of mammalian Puf proteins to target genes has not yet been reported. However, the expression of 13 genes that encode proteins involved in antibacterial and antifungal activity was upregulated in Drosophila Pum mutant, as determined by genome-wide analysis,¹³ although homologs of these genes do not exist in Arabidopsis.

Previous studies indicated that some Pufs localize predominantly to the cytoplasm, but diverse Pufs are also known to localize to multiple subcellular organelles.^14-16 CMV RNAs replicate in the vacuolar membranes and tonoplasts by means of replicase protein complexes.^17,18 In our study, some co-localization events were detected between APUM5-GFP and FM4-64 in the plasma membrane (Fig. 1A and B) and tonoplast-like structures (Fig. 1A and C). However, some of the regions labeled with APUM5-GFP did not coincide with FM4-64 labeling (Fig. 1A). In addition, some of the APUM5-GFP signal was detected in vesicle-like structures and in parts of the nucleus (Fig. 1D-F). Thus, APUM5 might have other functions to regulate endogenous target mRNAs because APUM5 localizes to multiple subcellular organelles.

Figure 1. Subcellular Localization of APUM5-GFP in N. benthamiana. (A) C-terminal GFP-tagged APUM5 was transiently expressed in N. benthamiana. The FM4-64 stain was used as a plasma membrane marker. Merged image shows colocalized regions. Scale bars are indicated at the pictures. (B) APUM5-GFP signals were merged with FM4-64 in the plasma membrane. (C) APUM5-GFP signals were merged with FM4-64 in vacuolar-like membrane. (Band C) Images are enlarged images of those indicated as boxes and circles in the (A) image. (D) APUM5-GFP was localized to vesicle-like structures and nucleus. The arrow indicates the nucleus. Red signals are chloroplast-auto signals. Scale bars are indicated at the pictures. (EandF) Enlarged images of those indicated by boxes in (D).

APUM5 bound to the putative Pumilio-binding motifs in the CMV RNAs and hbNRE2.¹⁹ Drosophila Pum binds to hbNRE2 mRNA and represses its ability to regulate embryo development via deadenlyase-dependent and -independent pathways.^20,21 This indicates that APUM5 might be associated with deadenylase-dependent and -independent pathways to repress CMV RNAs, even though CMV has a tRNA-like structure (TLS) instead of a poly(A) tail at its 3′ end.²² Interestingly, only CMV group-I but not CMV group-II strains contained conserved Pumilio-binding motifs at the 3′ UTR. Furthermore, some internal, putative Pumilio-binding motifs of CMV RNAs could also serve as targets for the repression of CMV infection (Fig. 2).

Figure 2. Proposed working model for APUM5 suppression of CMV infection. First, CMV entry into the host cells occurs by mechanical wounding or insects chewing. Then CMV RNAs replicate and synthesize viral proteins. CMV RNAs are stabilized by a tRNA-like structure (TLS) which is recognized by tRNA-specific enzymes. APUM5 recognizes the CMV 3′ UTR and some internal Pumilio binding motifs in the CMV genome either with unknown repression complexes or in association with Ccr4-Pop2p-NOT-like complex. Finally, APUM5 suppresses CMV infection via the direct binding of viral RNAs and attenuates CMV symptoms.

mRNA repression by yeast Puf3, 4 and 5, human Pum, Drosophila Pum and C. elegans FBF seems to follow a general regulatory mechanism.^4,23 For example, yeast Puf5 directly interacts with Pop2p and forms the Ccr4-Pop2p-NOT mRNA deadenylase complex that then attacks the 3′ UTR of target mRNA.^4,24 We investigated the Arabidopsis homologs of mammalian Pop2p deadenylase. Pop2p is also known as carbon catabolite repressor 4-CCR4-associated factor1 (CCR4-CAF1) and catalyzes mRNA deadenylation.²⁵ The CAF1 family in Arabidopsis has 11 members, some of which respond to environment and biotic stresses.²⁶ AtCAF1a and AtCAF1b have active involvement in biotic and abiotic stresses.²⁶ Thus, Arabidopsis APUM5 might interact with similar partners that are associated with deadenylation in planta through a more general model, as suggested by the Puf interaction with the Ccr4-Pop2p-NOT complex. Unlike Xenopus Pum2, APUM5 did not bind to the 5′ m7G cap structure for blocking the assembly of the initiation complex (Fig. 2).^19,27 Thus, APUM5 might inhibit CMV infection together with unknown repression complexes.

Puf protein is very useful to regulate target RNA because of sequence-specific binding capacity. PHD of Puf could be engineered to specifically recognize different Pumilio binding site in animal system.^28-30 However, plant Puf protein function is still not fully known compared with animal Pufs. Thus, more accurate analysis of the function of APUM5 and other APUM family needs to be done in plant. And further finding of its endogenous mRNA targets and de novo interacting partners could also be helpful in dissecting the function of plant Puf proteins.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.