Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina

Abstract

Background: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta.

Results: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24^+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24.

Conclusions: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3'-extended 5.8S pre-rRNA.

Keywords: Arabidopsis thaliana; Chara corallina; Charophyta; ITS2; Puf; RNA-binding proteins; rRNA.

Fig. 1

Phylogenetic trees of two nucleolar Pumilio protein families in the representative species of green plants (a and b) and the protein structures of nucleolar APUMs and ChPUMs (c). a and b Phylogenetic relationship among the putative nucleolar Pumilio proteins belonging to the APUM23 and ChPUM2 family (a) and the APUM24 and ChPUM3 family (b). The phylogenetic trees were constructed using the maximum likelihood LG + G model using MEGA7 software [29] with 1000 bootstrapping replicates. Two independent nucleolar Pumilio proteins of red algae (Chondrus crispus and Galdieria sulphuraria), Drosophila melanogaster, Homo sapiens, and Saccharomyces cerevisiae were used as outgroups. c Primary protein structures of APUM23 and APUM24 from Arabidopsis thaliana and ChPUM2 and ChPUM3 from Chara corallina. Black hexagons indicate Puf RNA-binding domains

Fig. 2

Amino acid sequence alignment of putative nucleolar Pumilio proteins, APUMs and ChPUMs. a Alignment of the amino acid sequences of APUM23 and ChPUM2. b Alignment of the amino acid sequences of APUM24 and ChPUM3. The Puf domains are indicated with black and gray lines above the amino acid sequences, and the five residues in the 2nd α-helix of each Puf domain that potentially interact with RNA bases are indicated with red boxes. The thin gray lines in the C-R5 Puf domain represent unfolded chains. Basic amino acids conserved in the N-terminal Puf domains of APUM24 family proteins are boxed in purple. Arrows under the purple box indicate the amino acids that are not conserved in ChPUM3. Conserved aromatic amino acids of APUM24 family proteins are boxed in green, and the basic amino acids in the C-terminal region are boxed in blue

Fig. 3

Predicted 3-D structures of putative nucleolar APUMs and ChPUMs. a C-shaped structures of APUM23 and ChPUM2. b L-shaped structures of APUM24 and ChPUM3. Unfolded side chains in the C-R5 domain are marked with red circles

Fig. 4

Nucleolar colocalization of APUM23-GFP and ChPUM2-RFP (upper panel) and APUM24-GFP and ChPUM3-RFP (lower panel) in N. benthamiana leaf cells. Scale bars = 50 μm

Fig. 5

Complementation assays of 35S:ChPUM2 and 35S:ChPUM3 transgenic plants in the apum23 mutant background. a Confirmation of the expression of ChPUM2 and ChPUM3 transgenes in the apum23–2 mutant using RT-PCR. Gel images were processed from original figures (Additional file 3: Figure S3) by Photoshop CS5. b Normal germination and root growth of apum23–2 complemented with 35S:ChPUM2.c Plant heights and rosette leaves in mature plants. Leaves were collected from 2-week-old plants. d Recovery of streptomycin susceptibility in the apum23–2 complemented 35S:ChPUM2.e qRT-PCR analysis for unprocessed rRNAs in wild-type Col-0, apum23–2, 35S:ChPUM2/apum23–2, and 35S:ChPUM3/apum23–2. Two technical and three biological replicates were performed for PCR measurements. Asterisks indicate the results of Student’s t-test between apum23–2 and transgenic plants (**; p < 0.01). Values represent means ± standard deviations, SDs (n = 3). The left panel shows a schematic diagram of poly(A) pre-rRNA byproducts and the primers used for the detection of pre-rRNAs. Unprocessed poly(A) 18S (~ 2.6 knt) and 5.8S (~ 300 nt) pre-rRNAs are shown below the 35S pre-rRNA

Fig. 6

Complementation assays of 35S:ChPUM2 and 35S:ChPUM3 transgenic plants in the apum24^+/− mutant background. a T-DNA insertion site of apum24–1 mutant alleles (upper panel) and genotyping (bottom panel). Primers used for genotyping are indicated with arrows. Original gel images for bottom panel are provided in Additional file 4: Figure S4a. b Confirmation of the expression of ChPUM2 and ChPUM3 transgenes in the apum24–1^+/− mutant using RT-PCR. Original gel images are provided in Additional file 4: Figure S4b. c Siliques of Col-0 control, apum24–1^+/−, and transgenic apum24–1^+/− expressing 35S:ChPUM2 or 35S:ChPUM3. The right panels for each plant line are enlarged images of the boxed regions. Arrows and arrowheads indicate undeveloped ovules and aborted seeds, respectively. Note that none of the transgenics complemented the abnormal seeds to normal levels. d qRT-PCR for analyzing relative unprocessed rRNA levels in Col-0 control, apum24–1^+/−, and 35:ChPUMN/apum24–1^+/− using the same primers that were used in Fig. 5. Two technical and three biological replicates were performed for PCR measurements. Values represent means ± SDs (n = 3) (**; p < 0.01)

Fig. 7

Restoration analyses of the salt- and sugar-sensitive apum23 and apum24 phenotypes by ChPUM2 and ChPUM3. aapum23–2 (left panel) and apum24–1^+/− (right panel) seedlings expressing either 35S:ChPUM2 or 35S:ChPUM3 in the absence or presence of NaCl and glucose. Col-0 Control (pB2GW7), apum24–1^+/−, 35S:ChPUM2/apum24–1^+/−, and 35S:ChPUM3/apum24–1^+/− seeds were germinated on 1/2 MS medium containing 10 mg L^− 1 Basta with the indicated treatment. b Expression levels of APUM24 in Col-0 Control (pB2GW7), apum24–1^+/−, and transgenic apum24–1^+/− plants expressing 35S:ChPUM2 and 35S:ChPUM3 in the absence and presence of 200 mM glucose. Note the similar expression levels of APUM24 in the presence of 200 mM glucose. Original gel images are provided in Additional file 5: Figure S5. c qRT-PCR analysis of the relative unprocessed rRNA levels in Col-0 Control (pB2GW7), apum24–1^+/−, and transgenic apum24–1^+/− plants expressing 35S:ChPUM2 and 35S:ChPUM3 in the presence of 200 mM glucose. The same primers were used as in Fig. 5. Two technical and three biological replicates were performed for PCR measurements. Values represent means ± SDs (n = 3).