The small nucleolar RNA SnoR28 regulates plant growth and development by directing rRNA maturation

Abstract

Small nucleolar RNAs (snoRNAs) are noncoding RNAs (ncRNAs) that guide chemical modifications of structural RNAs, which are essential for ribosome assembly and function in eukaryotes. Although numerous snoRNAs have been identified in plants by high-throughput sequencing, the biological functions of most of these snoRNAs remain unclear. Here, we identified box C/D SnoR28.1s as important regulators of plant growth and development by screening a CRISPR/Cas9-generated ncRNA deletion mutant library in Arabidopsis thaliana. Deletion of the SnoR28.1 locus, which contains a cluster of three genes producing SnoR28.1s, resulted in defects in root and shoot growth. SnoR28.1s guide 2'-O-ribose methylation of 25S rRNA at G2396. SnoR28.1s facilitate proper and efficient pre-rRNA processing, as the SnoR28.1 deletion mutants also showed impaired ribosome assembly and function, which may account for the growth defects. SnoR28 contains a 7-bp antisense box, which is required for 2'-O-ribose methylation of 25S rRNA at G2396, and an 8-bp extra box that is complementary to a nearby rRNA methylation site and is partially responsible for methylation of G2396. Both of these motifs are required for proper and efficient pre-rRNA processing. Finally, we show that SnoR28.1s genetically interact with HIDDEN TREASURE2 and NUCLEOLIN1. Our results advance our understanding of the roles of snoRNAs in Arabidopsis.

Figure 1

Mutations in the SnoR28.1 cluster result in pleiotropic developmental defects. A, Schematic diagram showing mutants generated by CRISPR/Cas9 for the SnoR28.1 cluster. B, RT-PCR showing the levels of SnoR180 and SnoR28.1s in wild-type and mutant seedlings. ACTIN2 was used as the control. C, Phenotypes of the aerial parts of wild-type and SnoR28.1 mutant seedlings. Scale bars: 5 mm. D, Schematic illustration of constructs containing snoR180-SnoR28.1a-SnoR28.1b-SnoR28.1c (C1), SnoR180 only (C2), SnoR180-SnoR28.1a (C3), SnoR180-SnoR28.1a-SnoR28.1b (C4), and SnoR28.1a-SnoR28.1b-SnoR28.1c (C5) driven by the endogenous NCR1 promoter, and SnoR28.1c (C6) driven by the 35S promoter. E, RT-PCR showing the levels of SnoR180 and SnoR28.1, with ACTIN2 as the control. F, Phenotypes of the aerial parts of wild type, ncr1, and the indicated transgenic seedlings. Scale bar: 5 mm. G, Phenotypes of the aerial parts of ncr1 and m9 plants complemented with SnoR28.1c (C6). RT-PCR of the levels of SnoR28.1c is shown below, with ACTIN2 as a loading control. Scale bar: 5 mm. See also Supplemental Figures S1–S3 and Supplemental Data Set S1.

Figure 2

SnoR28.1s are highly expressed in apical meristems and localize to the nucleolus. A, Expression profiles of SnoR28.1s in different tissues. The data are means ± SE. B, GUS staining showing the expression of pNCR1:GUS in different tissues. C, SnoR28.1 localizes to the nucleolus, as determined by FISH. FISH was performed using Antisense (AS) and sense (SE) probes for SnoR28.1. DAPI staining marks the nucleus. GFP-NUC1 marks the nucleolus. Scale bars: 2 μm. See also Supplemental Figure S4 and Supplemental Data Set S2.

Figure 3

SnoR28.1s are required for 2'-O-methylation of 25S rRNA at G2396. A, Schematic diagram of base pairing between the SnoR28.1a antisense box, extra box, and a region in 25S rRNA. Boxes C/D and C'/D' are shown in red, the antisense box and extra box are shown in blue, and substrate rRNAs are shown in yellow. Methylation site at 25S_G2396 is marked by an asterisk. B, Schematic diagram of the primer design for RTL-P. C, The detection of 2'-O-methylation at G2396 of 25S rRNA by RTL-P using different dNTP concentrations (1 μM and 1 mM). The relative levels of PCR products are shown. A high level of PCR products obtained with the MeUA primer under low dNTP conditions indicates a low level of methylation. D, Analysis of 2'-O-methylation level at G2396 of 25S rRNA by primer extension. Primer extension was performed in the presence of increasing dNTP concentrations (4 μM and 1 mM). A 32-nt 25S rRNA sequence from the primer to the methylated site was used as the marker. The position of the methylated site is marked with asterisks and an arrow. E, Changes in rRNA methylation at individual sites in the ncr1 mutant, as determined by RiboMeth-seq. The methylation sites in 25S rRNA are shown in black, the methylation sites in 18S rRNA are shown in blue, and the methylation sites in 5.8S rRNA are shown in red. 25S_G2396 is highlighted in yellow. See also Supplemental Figure S10 and Supplemental Data Set S3.

Figure 4

SnoR28.1s are required for the proper processing of pre-rRNA. A, SnoR28.1a interacts with 45S rRNA. Upper panel: Schematic diagram of pre-rRNA and the positions of the primer pairs (black bars) used for SnoR28.1a-ChIRP qRT-PCR. Lower panel: Affinity capture of SnoR28.1a by CHIRP. The SnoR28.1a probes detect SnoR28.1a. SnoR126 probes were used as negative controls (left). qRT-PCR detection of 45S rRNA regions that interact with SnoR28.1a (right). UBC21 and HID2 served as negative controls. Data represent mean ± SD (n = 4 independent experiments). B, Diagram illustrating the pre-rRNA processing intermediates that were detected by RNA gel blotting using the specific probes (p1–p4) highlighted with blue bars. C, RNA gel blots showing the levels of 35S, 32S, 27S (27SA or 27SB), and aberrant rRNA processing products 27SB* (blue) in the indicated seedlings. The SYBR Green II stained gel image is shown as a loading control. D, Identification of 27SB* in total RNA by circular RT-PCR. Asterisks (black and red) indicate the major aberrant processing products. E, Diagram of 27SB* cleavage sites. Arrowheads (black and red) indicate the positions of cutting sites corresponding to the major aberrant processing products in (D). See also Supplemental Figures S5–S7.

Figure 5

SnoR28.1 mutants have impaired ribosomal function. A, Polyribosome profiles of the indicated seedlings, as determined by measuring absorbance at 254 nm over a 15%–60% sucrose gradient. B, Identification of 27SB* in the polysome fraction by circular RT-PCR. Asterisks (black and red) indicate the aberrant processing products. C, Phenotypes of 10-d-old seedlings of the indicated genotypes grown vertically on ½ MS plates supplemented with different antibiotics. Scale bars: 1 cm. D, Quantification of root length of WT, ncr1, C1/ncr1, hid2, and ncr1 hid2 in (C). Asterisks indicate significant differences between the wild type and the indicated mutants (P < 0.05, n=7; one-way ANOVA along with Bonferroni’s multiple comparison, Supplemental Data Set S5).

Figure 6

Conserved motifs are important for the function of SnoR28.1s. A, Schematic diagram of different mutant forms of SnoR28.1a. B, Phenotypes of the aerial parts of ncr1 plants complemented with different mutant forms of SnoR28.1a. RT-PCR showing the expression levels of different mutant forms of SnoR28.1a is shown below, with ACTIN2 as a loading control. Scale bar: 5 mm. C, Primary root growth of 10-d-old seedlings of the indicated genotypes. Scale bar: 1 cm. D, Quantification of the primary root lengths in (C). In each boxplot, dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum root length, respectively. Different lowercase letters indicate statistically significant differences (P < 0.01; n = 24; one-way ANOVA along with Bonferroni’s multiple comparison, Supplemental Data Set S5). E, Analysis of the 2'-O-methylation levels at G2396 of 25S rRNA by primer extension in the wild type, ncr1, and the indicated transgenic seedlings. Primer extension was performed in the presence of increasing dNTP concentrations (4 μM and 1 mM). A 32-nt 25S rRNA sequence from the primer to the methylated site was used as the marker. The positions of the methylated site are marked with asterisks and arrowheads. F, Histogram plot with error bars showing variation in MethScore of 25S_G2396 between the wild type (n = 3) and the indicated mutants (n = 2 for ncr1, mut4, and mut5). Upward and downward histograms indicate MethScore greater and less than 0, respectively. P-value between the wild type and each mutant was calculated by single-tailed t test. G, RNA gel blot showing the levels of aberrant rRNA processing products 27SB* (blue) in wild type, ncr1, and the indicated transgenic seedlings. Probe p4 was used for RNA gel blot analysis. The SYBR Green II stained gel image is shown as a loading control. See also Supplemental Figure S8.

Figure 7

Genetic interactions of ncr1 with hid2 and nuc1. A, Phenotypes of 20-d-old wild-type, ncr1, hid2, and ncr1 hid2 double mutant seedlings grown on ½ MS plates. Scale bar: 5 mm. B, Primary root growth of 10-d-old seedlings of the indicated genotypes. Scale bar: 1 cm. C, Quantification of the primary root length in (B). In each boxplot, dark horizontal line, median; edges of boxes, 25th (bottom) and 75th (top) percentiles; whiskers, minimum and maximum root length, respectively. Different lowercase letters indicate statistically significant differences (P < 0.01, n = 25; one-way ANOVA along with Bonferroni’s multiple comparison, Supplemental Data Set S5). D, RNA gel blot showing the levels of aberrant rRNA processing products 27SB* (blue) in the indicated seedlings. Probe p4 was used for analysis. The SYBR Green II stained gel image is shown as a loading control. E, Phenotypes of 34-d-old plants of the indicated genotypes. Scale bar: 5 cm. See also Supplemental Figure S9.