Arabidopsis RIBOSOMAL RNA PROCESSING7 Is Required for 18S rRNA Maturation

Abstract

Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast rRNA processing protein 7 (Rrp7), which is required for 18S ribosomal RNA (rRNA) maturation. Arabidopsis rrp7 mutants exhibit a pleiotropic phenotype including slow growth, altered shoot phyllotaxy, aberrant venation in lateral organs, partial infertility, and abscisic acid hypersensitivity in seedlings. In Arabidopsis, RRP7 localizes mainly to the nucleolus, the site of the 45S rDNA transcription that produces a 45S pre-rRNA primary transcript, precursor of the 25S, 18S and 5.8S rRNAs. Lack of RRP7 function perturbs 18S rRNA maturation, causes nucleolar hypertrophy, and results in an increased 25S/18S rRNA ratio. Arabidopsis contains hundreds of 45S rDNA genes whose expression is epigenetically regulated, and deregulated, in rrp7 mutants. Double mutant analysis revealed synergistic interactions between RRP7 alleles and alleles of MAS2, NUCLEOLIN1 (NUC1), and HISTONE DEACETYLASE 6 (HDA6), which encode epigenetic regulators of 45S rDNA transcription. Our results reveal the evolutionarily conserved but divergent roles of RRP7 as a ribosome biogenesis factor.

Figure 1.

Structure of RRP7 and the Molecular Nature, Rosette Phenotype, and Transgene-Mediated Complementation of its Mutant Alleles. (A) Schematic representation of RRP7, including the positions and molecular nature of the rrp7 mutations. Black boxes represent exons and open boxes represent the 5′- and 3′-UTRs. Lines between boxes represent introns, and triangles represent T-DNA insertions. (B) to (J) Rosettes of Col-0, rrp7-1, and rrp7-2 plants as indicated. (K) to (M) Rosettes of rrp7-1 RRP7_pro:RRP7 (K), rrp7-2 RRP7_pro:RRP7 (L), and rrp7-2 35S_pro:RRP7:GFP (M) plants. Photographs were taken 7 (B) to (D), 14 (E) to (G) and (K) to (M), and 21 (H) to (J) days after stratification (DAS). Scale bars: 1 mm.

Figure 2.

Pleiotropic Morphological Phenotype of rrp7 Plants. (A) to (C) Presence of a single cauline leaf in the axils of Col-0 stems (A), and three cauline leaves in the axils of rrp7 mutant stems (B) and (C). (D) Adult Col-0 and rrp7-1 plants. (E) to (G) Dissected siliques from Col-0 (E), rrp7-1 (F), and rrp7-2 (G) plants. Red arrows indicate unfertilized ovules. (H) Fertility of rrp7 plants, expressed as the number of seeds per silique. Seeds from ten siliques collected from five plants per genotype were counted. (I) Flowering time (number of days from stratification to bolting) in the rrp7 mutants. (J) Number of rosette leaves that developed before bolting in rrp7 plants. The assays shown in (I) and (J) were performed with at least 30 plants of each genotype. Photographs were taken at 60 DAS (A) to (G). Error bars in (H) to (J) indicate standard deviations. Asterisks indicate values significantly different from the corresponding wild type in a Student′s t test (***p < 0.001). Scale bars = 1 cm (A) to (C); 3 cm (D); and 1 mm (E) to (G).

Figure 3.

Aberrant Venation Patterns in parl1-2 and rrp7-1 Cotyledons, Vegetative Leaves, and Petals. (A) to (L) Diagrams were drawn from micrographs taken from cotyledons (A), (E) and (I), first-node leaves (B), (F) and (J), third-node leaves (C), (G) and (K), and petals (D), (H) and (L). Cotyledons and leaves were collected at 21 DAS and petals were collected at 63 DAS from Col-0 (A) to (D), parl1-2 (E) to (H), and rrp7-1 (I) to (L) plants. Cotyledon, leaf and petal margins are shown in orange. Scale bars = 1 mm.

Figure 4.

Effect of Exogenous ABA on rrp7-1, smo4-3, parl1-2, and amiR-MAS2.1 Plants. Percentage of seedlings grown in medium supplemented with different concentrations of ABA that displayed green, fully expanded cotyledons when scored at 10 DAS. The abi4-2 and aba1-104 mutants were used as controls. The experiment was repeated three times, each with 156 seeds of each genotype, sown onto three different plates. Error bars indicate sd. Asterisks indicate values significantly different from the corresponding wild type in a Student′s t test (*P < 0.05, and **P < 0.01). The genetic background of abi4-2 and aba1-104 is Col-5, and that of all other mutants is Col-0.

Figure 5.

Subcellular Localization of RRP7. (A) to (C) Confocal laser-scanning micrographs of roots from plants homozygous for the RRP7_pro:RRP7:GFP transgene. Fluorescence signals correspond to Hoechst 33342 (A), GFP (B), and the merged image (C). (D) to (I) Immunolocalization of fibrillarin in plants homozygous for the RRP7_pro:RRP7:GFP transgene. Fluorescence signals show: fibrillarin detection in red (D) and (E); DAPI staining in blue (E) and (H); GFP fluorescence in green (G) and (H); and the corresponding merged images (F) and (I). Scale bars = 10 µm.

Figure 6.

Early Steps in 45S pre-rRNA Processing and 18S rRNA Maturation in the rrp7 Mutants. (A) Diagram illustrating the pre-rRNAs that can be detected in an RNA gel blot using the S7 and S9 probes. Modified from Hang et al. (2014). 5′-ETS and 3′-ETS, external transcribed spacers. ITS1 and ITS2, internal transcribed spacers. Red and green vertical stripes mark the regions of the pre-rRNAs to which the S7 and S9 probes hybridize, respectively. (B) and (C) RNA gel blots. Total RNA was separated in formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with the S9 (B) and S7 (C) probes. RNA was extracted from Col-0, rrp7-1, rrp7-2, and rrp7-1 RRP7_pro:RRP7 plants. EtBr: photographs of ethidium bromide-stained gels taken before blotting as loading controls. The 16S, 23Sa and 23Sb bands correspond to chloroplast rRNAs. A magnified view of the selected region is shown in (C), corresponding with a very short exposure time, which allows distinction between the 35S(P), and 33(P′)/32S pre-rRNAs. (D) Ethidium bromide-stained agarose gels (negative images) visualizing circular RT-PCR products. RNA was circularized and reverse transcribed using the 25SRT primer, and the cDNA obtained was PCR amplified with the r1+r2, r3+r2 and r4+r2 primer pairs. The cDNA PCR amplified with the r5+r6 primer pair was obtained from RNA circularized and reverse transcribed using the r1 primer.

Figure 7.

Subcellular Localization of 25S, 18S, and 5.8S rRNA Species in rrp7-1 Plants. (A) to (T) Fluorescence signals correspond to Hoechst 33342 (A), (H), (O) and (R), an 18S probe (B) and (I), a 25S probe (C) and (J), a 5.8S probe (F) and (M), acridine orange (P) and (S), and their corresponding merged images (D), (K), (G), (N), (Q) and (T). Scale bars = 10 µm.

Figure 8.

45S rDNA VAR Expression in smo4-3 and rrp7-1. (A) and (B) Schematic representation of the 45S pre-rRNA (A) and its 3′-ETS polymorphic region (B). 5′-ETS and 3′-ETS, external transcribed spacers. ITS1 and ITS2, internal transcribed spacers. The p3 and p4 primers were used for PCR amplifications (Supplemental Table 4). (C) to (E) PCR analysis of the relative abundance of 45S rDNA variants (VAR1-VAR4) in reverse-transcribed RNA (C), and genomic DNA (D) and (E), from Col-0, smo4-3, and rrp7-1 plants as indicated. Relative amounts of each 45S rDNA variant (E) were determined using the Agilent DNA 1000 kit on an Agilent 2100 Bioanalyzer. VAR4 was not detected. The ORNITHINE TRANSCARBAMYLASE (OTC) housekeeping gene (Quesada et al., 1999) was used as an internal control in (C).

Figure 9.

Genetic Interactions of rrp7-1 with mas2-1, smo4-3, parl1-2, nuc2-2, and hda6-7. (A) to (J) Phenotypes of rosettes of single (A) to (E) and double (F) to (J) mutants. Images show rosettes of mas2-1 (A), smo4-3 (B), parl1-2 (C), nuc2-2 (D), and hda6-7 (E) plants (top row) along with rrp7-1 mas2-1 (F), rrp7-1 smo4-3 (G), rrp7-1 parl1-2 (H), rrp7-1 nuc2-2 (I), and rrp7-1 hda6-7 (J) plants (bottom row). Photographs were taken at 21 DAS. Scale bars = 1 mm.

Figure 10.

Proposed Roles for RRP7 in 45S pre-rRNA Processing Based on the Molecular Phenotype of the rrp7 Mutants. Red arrows indicate over-accumulated (↑↑↑) or depleted (↓) pre-rRNAs, as detected by RNA gel blots and circular RT-PCR assays using the rrp7 mutants. In our model, Arabidopsis RRP7 is assumed to participate in the steps of 45S pre-rRNA processing in which the substrate is overaccumulated in the rrp7 mutants (P′, P₁ and C₂ cleavages; orange ovals). The question mark in C₂ cleavage (bottom middle) indicates that overaccumulation of 27SB pre-rRNA can alternatively be explained as an indirect effect of impaired 18S rRNA production. RRP7 might also participate in the steps in which the product is depleted in the rrp7 mutants (P₂ cleavages; gray ovals). Because RRP7 participates in the P′ cleavage at the 5′-ETS-first pathway, it might also cleave the same site at the ITS1-first pathway (yellow oval). Short-lived pre-rRNAs are shown in faint colors. Other details are as shown in Supplemental Figure 2.