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.

SnoR28, a conserved box C/D small nucleolar RNA cluster, regulates plant growth and development by guiding 2′-O-methylation of 25S rRNA at G2396 and facilitating proper and efficient pre-rRNA processing.

IN A NUTSHELL.

Background: Small nucleolar RNAs (snoRNAs) are a class of abundantly expressed noncoding RNAs (ncRNAs) that guide modifications of ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs) within the nucleolus. In plants, many snoRNAs have been identified and annotated. However, the biological functions of most snoRNAs in higher plants remain unclear. Based on sequences of conserved elements, snoRNAs are classified into box C/D or box H/ACA snoRNAs, which catalyze 2'-O-ribose methylation and pseudouridinylation, respectively. SnoRNAs associate with different sets of core proteins to form small nucleolar ribonucleoprotein particles. For rRNA modification, they base pair with complementary sequences on rRNA precursors to achieve site-specificity. In addition to rRNA modification, a few snoRNAs are required for proper and efficient pre-rRNA processing.

Question: What is the function of Arabidopsis thaliana snoRNAs whose functions remain to be characterized? Can we use a reverse genetic approach to identify snoRNAs with important functions in plant development?

Findings: By screening a library of CRISPR/Cas9-generated ncRNA deletion mutants, we identified SnoR28.1, a locus containing a cluster of genes producing conserved box C/D snoRNAs, as an important regulator of plant growth and development. Deletion of the SnoR28.1 cluster results in pleiotropic root and shoot growth defects. SnoR28.1s guide 2'-O-ribose methylation of 25S rRNA G2396 and facilitate pre-rRNA processing, which is essential for proper ribosome function. SnoR28 contains a 7-bp antisense box (shortest) and an 8-bp “extra box” that is complementary to the rRNA methylation site nearby. The antisense box is required for 25S rRNA methylation and pre-rRNA processing. The “extra box” is also required for pre-rRNA processing, but is partially responsible for methylation. Our results provide insight into the biological role of a snoRNA cluster in plants.

Next steps: We are using ribosome profiling to identify proteins whose translation is affected by loss of SnoR28.1s. The biological importance of other snoRNAs in plants also remains an exciting question for future research.

Introduction

Noncoding RNAs (ncRNAs), which lack protein-coding potential, account for the majority of RNAs in eukaryotic cells (Wright and Bruford, 2011). ncRNAs include housekeeping ncRNAs such as transfer RNAs (tRNAs; Wright and Bruford, 2011), ribosomal RNAs (rRNAs; Hillis and Dixon, 1991; Saez-Vasquez and Delseny, 2019), small nuclear RNAs (snRNAs; Sontheimer and Steitz, 1993; Matera et al., 2007), and small nucleolar RNAs (snoRNAs; Bachellerie et al., 2002; Decatur and Fournier, 2003), as well as regulatory ncRNAs such as microRNAs (miRNAs; Bartel, 2009; Shukla et al., 2011), small interfering RNAs (siRNAs; Elbashir et al., 2001; Dorsett and Tuschl, 2004), and long noncoding RNAs (lncRNAs; Mercer et al., 2009). Housekeeping ncRNAs play key roles in basic biological processes, while regulatory ncRNAs are important for the regulation of gene expression.

SnoRNAs guide modifications of rRNAs, tRNAs, and snRNAs within the nucleolus (Bachellerie et al., 2002; Kiss, 2002; Decatur and Fournier, 2003). Based on the presence of conserved nucleotide sequences, snoRNAs are classified as box C/D snoRNAs or box H/ACA snoRNAs (Balakin et al., 1996; Bachellerie et al., 2002). Box C/D snoRNAs generally contain box C (RUGAUGA, where R is a purine) and D (CUGA) motifs near the 5′ and 3′ ends, respectively, and the related C′ and D′ motifs in the internal region (Vidovic et al., 2000; Watkins et al., 2000; Bachellerie et al., 2002; Nolivos et al., 2005). Box H/ACA snoRNAs contain an H box (ANANNA, where N is any nucleotide) and an ACA box (Ganot et al., 1997; Bachellerie et al., 2002). Box C/D snoRNAs guide 2′-O-ribose methylation (2′-O-methylation; Kiss-Laszlo et al., 1996; Lowe and Eddy, 1999; van Nues et al., 2011), while box H/ACA snoRNAs guide pseudouridinylation (Kiss-Laszlo et al., 1996; Ni et al., 1997).

The C and D boxes, which are highly conserved in all C/D box snoRNAs, form a kink-turn structure through nonconventional base pairing (Bachellerie et al., 2002). The box C/D core motif directs the binding of four common core proteins, including the RNA binding protein Snu13 (15.5K), the methyltransferase fibrillarin (FIB), and two scaffolding proteins, Nop56 and Nop58, to target rRNAs (Bachellerie et al., 2002; Kiss, 2002; Decatur and Fournier, 2003). To guide rRNA methylation, the region adjacent to the D or D′ box in the snoRNA base pairs with rRNA, and the fifth nucleotide upstream of the D or D′ box in the rRNA is targeted for modification (the D + 5 rule; Bachellerie et al., 2002). Analysis of box C/D snoRNAs from yeast and human revealed additional conserved sequences in many snoRNAs that are complementary to regions adjacent to the rRNA methylation site; this “extra base-pairing” can stimulate rRNA methylation (van Nues et al., 2011). In addition to guiding methylation, a few box C/D snoRNAs participate in pre-rRNA processing and ribosome assembly. For instance, the box C/D snoRNAs U3, U8, and U14 are required for rRNA processing (Kass et al., 1990; Qu et al., 1995; Peculis, 1997; Lange et al., 1998; Borovjagin and Gerbi, 1999; Sharma and Tollervey, 1999).

In Arabidopsis thaliana, 118 different genes and 214 gene variants of box C/D snoRNAs and 72 different genes and 104 gene variants of box H/ACA snoRNAs have been identified by computational prediction and analysis of sequencing data (Wu et al., 2021). Many plant snoRNAs and their targets are well-conserved throughout the plant kingdom (Patra Bhattacharya et al., 2016). Plant snoRNA genes are often arranged as gene clusters (Barneche et al., 2001; Qu et al., 2001; Brown et al., 2003). The clustered snoRNA genes are transcribed polycistronically as a precursor RNA, which is then processed into individual snoRNAs. While most plant snoRNAs localize to the nucleus, a few snoRNAs localize to the cytosol, implying that these snoRNAs have alternative functions (Streit et al., 2020). HIDDEN TREASURE 2 (HID2), a box C/D snoRNA, promotes seed germination, root and seedling growth, and development (Zhu et al., 2016). HID2 does so by associating with the precursor 45S rRNA and increasing the efficiency and accuracy of pre-rRNA processing (Zhu et al., 2016). However, the roles of most snoRNAs in regulating plant growth and development and the underlying regulatory mechanisms are unknown.

In this study, by performing a forward genetic screening for ncRNAs that regulate plant growth and development, we found that SnoR28.1, a conserved locus containing a cluster of three box C/D snoRNA genes, is required for normal plant growth and development. SnoR28 guides 2′-O-methylation of 25S rRNA at G2396 and is required for proper and efficient rRNA processing and ribosome function. Our results provide insights into the biological role of a snoRNA cluster in plants.

Results

Deletion of a SnoR28.1 gene cluster results in growth and developmental defects in Arabidopsis

To identify ncRNAs that are important for plant growth and development, we constructed a library of 57 mutants for different ncRNAs using the CRISPR/Cas9 system (Supplemental Data Set S1). One of the mutants, referred to as noncoding RNA 1 (ncr1), exhibited pleiotropic developmental defects, including delayed germination, development of narrow first true leaves with pointed tips, retarded root growth, and a delayed transition to the reproductive phase (Supplemental Figure S1, A–C). Two independent ncr1 lines, which were isolated from different T1 transgenic plants, showed the same developmental defects (Supplemental Figure S1B). Since these lines harbor exactly the same mutation at the NCR1 (AT5G02645) locus, we used one of these lines for subsequent experiments.

The ncr1 mutant harbors a deletion of SnoR180, SnoR28.1a, and SnoR28.1b; these genes are organized in a polycistronic gene cluster (Figure 1A). As expected, SnoR180, SnoR28.1a, and SnoR28.1b were not expressed in ncr1 (Figure 1B). The deletion also interferes with the expression of downstream SnoR28.1c, as SnoR28.1c could not be detected (Figure 1B). We also examined the expression levels of adjacent genes, including AT5G17240, AT5G17250, AT5G17260, and AT5G17270, in ncr1 and found that their expression levels were comparable in the wild type and ncr1 (Supplemental Figure S1D). Complementation of ncr1 with a genomic DNA fragment encompassing SnoR180, SnoR28.1a, SnoR28.1b, and SnoR28.1c (C1) under the control of their endogenous promoter (2 kb of genomic sequence upstream of NCR1) completely rescued the phenotypes of ncr1 (Figure 1, D–F). Our results suggest that the pleiotropic developmental defects exhibited by ncr1 are caused by the loss of SnoR180, SnoR28.1a, SnoR28.1b, and SnoR28.1c.

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.

To pinpoint the snoRNA whose loss is responsible for the phenotype observed in ncr1, we deleted these four snoRNA genes individually and in combination using the CRISPR-Cas9 system (Figure 1, A and B; Supplemental Figure S2A), generating the mutants m2 (SnoR180 deleted), m3 (SnoR28.1a deleted), m4 (SnoR28.1b deleted), m5 (SnoR28.1c deleted), m6 (SnoR180 and SnoR28.1a deleted), m7 (SnoR28.1a and SnoR28.1b deleted), m8 (SnoR28.1b and SnoR28.1c deleted), and m9 (SnoR28.1a, SnoR28.1b, and SnoR28.1c deleted). Only the m9 mutant was phenotypically similar to ncr1, while all other mutants (m2 to m8) were phenotypically normal (Figure 1C; Supplemental Figure S2, B and D). We complemented the ncr1 mutant with genomic DNA fragments encompassing SnoR180 (C2), SnoR180 and SnoR28.1a (C3), SnoR180, SnoR28.1a, and SnoR28.1b (C4), and SnoR28.1a, SnoR28.1b and SnoR28.1c (C5), respectively, under the control of their endogenous promoter (Figure 1, D and E). C2 was unable to rescue the phenotypes of ncr1, whereas C3, C4, and C5 rescued the phenotypes of this mutant (Figure 1F; Supplemental Figure S2, C and E). We also complemented ncr1 and m9 with SnoR28.1c (C6) driven by the 35S promoter (Figure 1D) and found that C6 was able to fully rescue the phenotypes of ncr1 and m9 (Figure 1G). These results suggest that the SnoR28.1 cluster is important for plant growth and development and that the three snoRNAs in this cluster are functionally redundant.

SnoR28.1s are preferentially expressed in metabolically active tissues

To delineate the role of the SnoR28.1 cluster in plant development, we measured the levels of SnoR28.1a, SnoR28.1b, and SnoR28.1c in different tissues (seed, root, rosette leaf, stem leaf, stem, and flower) by performing RT-qPCR. These three snoRNAs were most abundant in seeds, seedlings, and flowers (Figure 2A). We also transformed wild-type plants with a β-glucuronidase (GUS) reporter gene whose expression was driven by 2 kb of the genomic sequence upstream of the SnoR28.1 cluster. GUS activity was enriched in the shoot apex, root tip, and seed (Figure 2B). Overall, our results indicate that SnoR28.1s are preferentially expressed in metabolically active tissues and seeds of Arabidopsis.

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.

SnoR28s are typical box C/D snoRNAs

SnoR28s were previously annotated as box C/D snoRNAs (Brown et al., 2003; Patra Bhattacharya et al., 2016; Wu et al., 2021). The Arabidopsis genome has five SnoR28 genes belonging to two distinct clusters (Supplemental Figure S3A). The SnoR28.1 cluster includes SnoR28.1a (94-nt), SnoR28.1b (96-nt), and SnoR28.1c (97-nt). The SnoR28.2 cluster includes SnoR28.2a (100-nt) and SnoR28.2b (103-nt). Sequence alignment showed that all of these sequences contain a conserved box C (5′-UGAUGA-3′), box D (5′-CUGA-3′), and an antisense box that is complementary to the rRNA target and used to guide 2′-O-methylation of the rRNA target (Wu et al., 2021). SnoR28s also contain C′ and D′ motifs in the internal region (Supplemental Figure S3A). SnoR28.1 levels were high in metabolically active tissues, but SnoR28.2s were not detected in seedlings under normal growth conditions (Supplemental Figure S3B). However, overexpressing the SnoR28.2 cluster fully rescued the phenotypes of ncr1 (Supplemental Figure S3C), indicating that SnoR28.2s are functional.

To explore the molecular function of SnoR28.1s, we performed fluorescence in situ hybridization (FISH) to examine the cellular localization of the three snoRNAs and validated that they localized to the nucleolus (Figure 2C). Next, to determine whether SnoR28.1s form small nucleolar ribonucleoprotein particles (snoRNPs) by associating with evolutionarily conserved proteins that are essential for rRNA methylation, we performed an RNA pulldown–mass spectrometry assay in vitro and identified conserved proteins in snoRNPs (including FIB1, FIB2, NOP56-like proteins, NOP58-like proteins, and SNU13-like proteins) and NUCLEOLIN1 (NUC1; Supplemental Figure S4A and Supplemental Data Set S2). NUC1 localizes to the nucleolus and is involved in rRNA processing, ribosome biosynthesis, and plant development (Saez-Vasquez et al., 2004b; Kojima et al., 2007; Pontvianne et al., 2010; Durut et al., 2014). The interaction between SnoR28.1s and NUC1 was confirmed by RNA immunoprecipitation (RIP) experiments (Supplemental Figure S4B). Together, these results validate the finding that SnoR28.1s are typical box C/D snoRNAs.

SnoR28.1s are required for 2′-O-methylation of 25S rRNA at G2396

SnoR28.1s were predicted to guide the methylation of G2396 of 25S rRNA (Wu et al., 2021). The antisense box mediates a 7-bp base-pairing interaction between SnoR28.1s and 25S rRNA (Figure 3A). We detected an 8-bp extra base-pairing mediated by an extra box (Figure 3A), which may reinforce the binding of SnoR28.1s with 25S rRNA. To investigate whether SnoR28.1s indeed guide the methylation of 25S rRNA at G2396, we performed reverse transcription at low deoxy-ribonucleotide triphosphate concentrations followed by PCR (RTL-P) in the wild-type and ncr1 mutant. In this experiment, extension of the site-specific MeUA-RT primer by reverse transcriptase is impeded by a 2′-O-methyl group at low dNTP concentrations, resulting in a lower abundance of PCR products, whereas extension of the MeA-RT primer is unaffected regardless of the dNTP concentration (Figure 3B). The MeUA-RT PCR products were less abundant than the MeA-RT PCR products at a low dNTP concentration in the wild type (Figure 3C), suggesting that 2′-O-methylation of 25S rRNA at G2396 occurs in the wild type. By contrast, the MeUA-RT PCR products were as abundant as the MeA-RT PCR products at a low dNTP concentration in ncr1 (Figure 3C), suggesting a loss of 2′-O-methylation of 25S rRNA at G2396 in ncr1. The abundance of the MeUA-RT PCR products in m9 and C5-complemented ncr1 (C5/ncr1) was comparable to that in ncr1 and the wild type, respectively (Figure 3C), suggesting that 2′-O-methylation of 25S rRNA at G2396 is guided by SnoR28.1s.

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.

To further confirm the impact of disrupting the SnoR28.1 gene cluster on the methylation of 25S_G2396, we performed primer extension at different dNTP concentrations (Dong et al., 2012). In primer extension, the passage of reverse transcriptase is blocked by 2′-O-methylated nucleotides in the RNA template at low dNTP concentrations, resulting in the appearance of short extension products, which is attenuated at higher dNTP concentrations. We detected a stop in reverse transcription in the wild type and C5/ncr1 in the presence of 4 μM dNTPs, which was not detected in ncr1 or m9 (Figure 3D). These results confirm the notion that 2′-O-methylation of 25S rRNA at G2396 is dependent on the SnoR28.1 cluster.

To examine whether SnoR28.1s also guide rRNA methylation at other sites, we performed RiboMeth-seq to profile 2′-O-methylation on rRNA in the wild type and ncr1. Only the methylation level at the 25S_G2396 site was significantly reduced in ncr1 (Figure 3E; Supplemental Data Set S3). Together, these results indicate that SnoR28.1s are specifically required for 2′-O-methylation of 25S rRNA at G2396.

SnoR28.1 is required for proper and efficient pre-rRNA processing in Arabidopsis

Besides guiding site-specific 2′-O-methylation on rRNA, box C/D snoRNAs may also regulate pre-rRNA processing (Borovjagin and Gerbi, 1999; Sharma and Tollervey, 1999; Zhu et al., 2016). 18S rRNA, the RNA component of the small (40S) ribosomal subunit, and 5.8S and 25S/28S rRNAs, two RNA components of the large (60S) ribosomal subunit, are processed from a long pre-rRNA, in which mature 18S, 5.8S, and 25S rRNAs are separated by internal transcribed spacers 1 (ITS1) and ITS2 and flanked by 5′ external transcribed spacers (5′-ETS) and 3′-ETS (Saez-Vasquez and Delseny, 2019). To explore whether SnoR28.1s participate in the regulation of pre-rRNA processing, we first examined whether SnoR28.1s are associated with 45S pre-rRNA by performing chromatin isolation by RNA purification (ChIRP). Our results revealed that 45S pre-rRNA could be retrieved by SnoR28.1 in ChIRP (Figure 4A), suggesting that SnoR28.1s are associated with 45S pre-rRNA.

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.

We then performed RNA gel blotting using probes targeting different regions to determine the steady-state levels of intermediate RNAs during pre-rRNA processing (Zhu et al., 2016). Probe p1, which targets 5′-ETS, distinguished the 35S/33S precursors from the putative 32S rRNA. p2, which targets ITS1, specifically detected 18S precursors, including P-A3, P′-A3, and 18S-A3 in the pre-40S small subunit. p3 in ITS1, and p4 in ITS2 were used to detect the variants of 27S fragments (27SA and 27SB) and pre-5.8S rRNAs in the pre-60S large subunit (Figure 4B). The ncr1 mutant accumulated more 35S/33S precursors, putative 32S rRNA, and 27SA/27SB variants compared to the wild type (Figure 4C). Multiple bands, perhaps corresponding to unknown intermediate RNAs, were also detected in ncr1 when probe p4 was used (Figure 4C, asterisk). Similar to ncr1, the m9 mutant accumulated known and unknown intermediate RNAs (Figure 4C). The accumulation of known and unknown intermediate RNAs in ncr1 was abolished when ncr1 was complemented with the C5 construct (Figure 4C). Our results suggest that the loss of SnoR28.1s causes the accumulation of intermediate RNAs in ncr1 and m9.

To identify the nature of the unknown bands, we performed circular RT-PCR to amplify misprocessed 25S rRNAs using polysomal RNA as a template and primers specific to the 5′-end of mature 25S rRNA (Supplemental Figure S5A). Misprocessed 25S rRNAs could be amplified in ncr1 and m9, but not in the wild type. Interestingly, more misprocessed 25S rRNAs were detected in ncr1 and m9 than in hid2 (Figure 4D). We submitted the misprocessed 25S rRNAs for sequencing. Our sequencing results revealed that the unknown intermediate RNAs in ncr1 were a series of 3′-truncated forms of 27SB (abbreviated as 27SB*) that are produced by cleavage within the 25S region (Figure 4E; Supplemental Figure S5B).

To determine whether the efficiency of pre-rRNA processing is affected in ncr1, we measured the levels of different pre-rRNA variants (Supplemental Figure S6A) in this mutant. These variants differ in their 3′-ETS sequences and their temporal and spatial expression patterns, as observed in various wild-type Arabidopsis accessions (Pontvianne et al., 2010; Micol-Ponce et al., 2018; Saez-Vasquez and Delseny, 2019). Pre-rRNA Variant 2 and Variant 3 accumulated to higher levels in ncr1 than in the wild type (Supplemental Figure S6B). This overaccumulation was largely abolished in ncr1 complemented with C1 (Supplemental Figure S6B). To determine whether the overaccumulation of Variant 2 and Variant 3 was attributed to increased pre-rRNA transcription, we examined whether SnoR28s could bind rDNA by performing ChIRP-qPCR. SnoR28.1a was unable to bind rDNA, excluding the possibility that SnoR28.1s promote pre-rRNA transcription to increase the accumulation of pre-rRNAs (Supplemental Figure S7). Together, our results suggest that the efficiency of pre-rRNA processing is reduced in ncr1 and it is compromised by the loss of SnoR28.1s.

SnoR28.1 is required for normal ribosome function in Arabidopsis

To explore the ribosome assembly state in ncr1, we examined the polyribosome profile in ncr1 by measuring the absorbance at 258 nm after traditional biochemical purification of ribosomes and sucrose density gradient sedimentation. Compared to the wild type, the ncr1 mutant had an increased ratio of polyribosomes (Figure 5A). This imbalanced polysome profile was restored to normal in C5/ncr1 (Figure 5A), indicating that the SnoR28.1 cluster regulates ribosomal assembly. Interestingly, 27SB* was detected in polysome fractions from the ncr1 mutant through circular RT-PCR (Figure 5B), indicating that the overaccumulation of 27SB* may lead to the abnormal ratio of polyribosomes in ncr1.

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).

To further determine whether ribosome function was affected in the ncr1 mutant, we tested the resistance of ncr1 to several aminoglycoside antibiotics, including gentamicin, streptomycin, kanamycin, spectinomycin, and erythromycin (Rosado et al., 2010; Hang et al., 2014; Zhu et al., 2016). These aminoglycoside antibiotics strongly affect translation by targeting the acceptor site (A site) in the ribosome during translation (Wilson, 2014). Chloramphenicol, which prevents protein chain elongation by blocking peptidyl transferase activity (Spahn and Prescott, 1996), was used as a negative control. The ncr1 mutant developed longer roots and cotyledons than the wild type when treated with gentamicin, streptomycin, kanamycin, spectinomycin, and erythromycin (Figure 5, C and D). However, the ncr1 mutant was not different from the wild type in terms of sensitivity to chloramphenicol (Figure 5C). The reduced sensitivity of ncr1 to aminoglycoside antibiotics was restored to normal in the complemented line C5/ncr1 (Figure 5, C and D). These observations strongly suggest that the ncr1 mutant contains a population of abnormal ribosomes that are less or not at all targeted by aminoglycoside antibiotics.

SnoR28s contain conserved motifs that are important for their function

A sequence comparison revealed that the motifs identified in SnoR28s, including the C/D box, C′/D′ box, antisense box, and extra box, are conserved in rice (Oryza sativa), Medicago truncatula, maize (Zea mays), zebrafish (Danio rerio), mouse (Mus musculus), and human (Homo sapiens; Supplemental Figure S8A). Furthermore, the expression of rice SnoR28.3 fully rescued the phenotype of ncr1 (Supplemental Figure S8, B–G). These results indicate that the functions of SnoR28s are conserved. We then determined which boxes, including box C, box D, the antisense box, and the extra box, are important for the function of SnoR28s. We generated five constructs, mut1 to mut5, which produce different mutant forms of SnoR28.1a (Figure 6A). The mut1 and mut2 constructs produce SnoR28.1a containing a mutated box C and box D sequence, respectively. The mut3 construct produces SnoR28.1a, whose nucleotides in the antisense box were all mutated to A. The mut4 construct produces a SnoR28.1a variant that only contains a C to A substitution in the antisense box (which is complementary to 25S_G2396; Figure 3A). The mut5 construct produces a SnoR28.1a variant that contains five conserved nucleotides in the extra box mutated to A. These constructs were transformed into ncr1 individually. The mut1 to mut4 constructs failed to rescue the developmental defects of ncr1 (Figure 6, B–D), suggesting that each of the classical motifs in SnoR28.1s is essential for the normal growth and development of Arabidopsis. Consistent with the growth phenotypes of the transgenic plants, the mut1-mut4 constructs failed to rescue the rRNA methylation and pre-rRNA processing defects of ncr1 (Figure 6, E–G). Unexpectedly, the mut5 construct partially rescued the growth and developmental defects of ncr1 (Figure 6, B–D). Interestingly, mut5 partially rescued 2′-O-methylation on 25S_G2396 (77.6%) in ncr1, but it did not rescue the defective pre-rRNA processing in ncr1 (Figure 6, E–G). These results suggest that the extra box helps guide 2′-O-methylation but plays an important role in pre-rRNA processing.

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.

SnoR28.1s synergistically interact with HID2 and NUC1

The developmental abnormalities of ncr1 resemble those of hid2 (Zhu et al., 2016). To explore the genetic interacting between ncr1 and hid2, we obtained the ncr1 hid2 double mutant by crossing. The ncr1 hid2 mutant exhibited slower leaf development and shorter roots (Figure 7, A–C) than ncr1 or hid2, suggesting that ncr1 and hid2 have an additive effect on the growth and development of Arabidopsis. RNA gel blotting showed that more 27SB* accumulated in ncr1 hid2 compared to ncr1 or hid2 during pre-rRNA processing (Figure 7D). In addition, the ncr1 hid2 double mutant developed longer roots and greener cotyledons than ncr1 or hid2 under streptomycin and erythromycin treatment (Figure 5C), indicating that the combination of ncr1 and hid2 increases plant resistance to streptomycin and erythromycin. Thus, ncr1 and hid2 have an additive effect on ribosome function.

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.

NUC1 is involved in 45S pre-rRNA processing (Saez-Vasquez et al., 2004a, 2004b). Since SnoR28.1s are associated with NUC1 (Supplemental Figure S4), we generated the ncr1 nuc1 double mutant to test the genetic interaction between these two genes. The ncr1 nuc1-2 double mutant showed an additive phenotype, such as smaller leaves and shorter plants compared to the single mutants (Figure 7E; Supplemental Figure S9), indicating that SnoR28.1s genetically interact with NUC1.

Discussion

In this study, we experimentally determined that SnoR28.1a, SnoR28.1b, and SnoR28.1c, produced by the SnoR28.1 cluster, redundantly regulate plant growth and development. These snoRNAs were determined to guide 2′-O-methylation of 25S rRNA at G2396. Furthermore, they promote efficient pre-rRNA processing to ensure proper ribosome assembly and function.

In the m2 to m8 mutants, at least one member of the SnoR28.1s is expressed (Figure 1). These mutants develop normally, suggesting that the expression of any SnoR28.1 is sufficient to fulfill the role of SnoR28.1s. Thus, SnoR28.1s are functionally redundant in plant growth and development. In our complementation experiments, the C2 construct, which only encodes SnoR180, failed to rescue the phenotypes of ncr1 (Figure 1, D–F), suggesting that these phenotypes are not caused by the loss of SnoR180. The C3 to C6 constructs, which encode at least one member of the SnoR28.1s, fully rescued the ncr1 phenotypes (Figure 1, D–G), also suggesting SnoR28.1s are functionally redundant in plant growth and development.

Chemical modifications of rRNA are crucial for the assembly and function of ribosomes (Lafontaine, 2015). We found that SnoR28.1s are required for 2′-O-methylation of 25S rRNA at G2396. G2396 is located in domain V of 25S rRNA, which lies in the subunit interface of the 70S/80S ribosome (Azevedo-Favory et al., 2021). This interface contains the peptidyl-transferase center, which is responsible for peptidyl-transferase activity and tRNA binding (Noller, 1991; Chattopadhyay et al., 1996; Biedka et al., 2018). In yeast, the loss of methylation on 25S rRNA alters the structure of 25S rRNA, ultimately resulting in dramatic ribosome instability (Gigova et al., 2014). In light of this observation, we propose that in the absence of G2396 methylation, domain V of 25S rRNA is improperly structured and a population of ribosomes forms abnormally in the ncr1 mutant. Aminoglycoside antibiotics such as streptomycin inhibit the binding of aminoacyl tRNAs to A- and P-sites in the ribosome (Wilson, 2014). The ncr1 mutant is resistant to streptomycin, supporting the notion that the ribosomal structure is altered in ncr1, which prohibits the proper binding of antibiotics. Overall, these results suggest that the loss of an rRNA modification strongly affects the structures of large ribosomal complexes.

The extra pairing adjacent to the target site between the extra box of box C/D snoRNAs and rRNA was previously shown to stimulate rRNA methylation in humans and yeast (van Nues et al., 2011). A recent genome-wide profiling of RNA ribose methylation in Arabidopsis revealed that approximately half of box C/D snoRNAs undergo extra pairing with nearby sequences of methylation sites (Wu et al., 2021). However, the function of extra pairing in plants is still unclear. The SnoR28/25S rRNA interaction entails a 7-bp major pairing and an 8-bp extra pairing (Figure 3). Our findings reveal that the extra pairing is also involved in SnoR28-mediated rRNA methylation on 25S_G2396, since the extra-box mutant (mut5) did not fully rescue the methylation on 25S_G2396 in ncr1 (Figure 6F). Our findings suggest that the extra pairing might facilitate snoRNA targeting and stimulate rRNA methylation in plants.

While most box C/D snoRNAs function in rRNA methylation, a subset of these snoRNAs are involved in pre-rRNA processing. In plants, U3 forms a stable complex with NUC1 and directly binds nascent pre-rRNA at the 5′-ETS and specifically cuts pre-rRNA at the P sites (Saez-Vasquez et al., 2004a). HID2 monitors efficient pre-rRNA processing by directly interacting with 45S pre-rRNA (Zhu et al., 2016). We demonstrated that SnoR28.1s are also required for efficient and proper pre-rRNA processing. SnoR28.1s directly interact with 45S pre-RNA. Moreover, both the major pairing and extra pairing are required for pre-rRNA processing, suggesting that the interaction between SnoR28.1s and pre-rRNA is important for processing. Although multiple snoRNAs have been implicated in rRNA processing, their precise mechanisms in regulating this process remain to be investigated. It would be interesting to determine whether SnoR28-dependent 2′-O-methylation is required for rRNA processing or if they are not coupled.

Deletion of the SnoR28.1 cluster results in pleiotropic defects in root and shoot growth. 25S rRNA methylation, rRNA processing, and ribosome assembly and function were found to be impaired in the absence of the SnoR28.1 cluster. These impairments may affect the translation of some proteins, leading to the growth defects displayed by ncr1. It would be interesting to identify proteins whose translation is regulated by SnoR28.1s by performing comparative ribosome sequencing analysis of the wild type and ncr1. In Arabidopsis, HID2-regulated rRNA biogenesis is also required for the proper assembly and activity of the ribosome (Zhu et al., 2016). Interestingly, the ncr1 hid2 double mutant showed more serious growth and developmental defects than the single mutants, suggesting these two C/D box snoRNAs have a cumulative and synergistic effect on ribosome function. In this study, we only characterized a few developmental phenotypes of the ncr1 single and ncr1 hid2 double mutants that are manifested under normal growth conditions. Since SnoR28 and HID2 are required for the proper function of ribosomes, we speculate that these snoRNAs may be involved in additional aspects of plant development and may also be involved in stress responses.

Our results show that SnoR28 interacts with NUC1 (Supplemental Figure S4), and the ncr1 nuc1-2 double mutant showed an additive phenotype (Figure 7E; Supplemental Figure S9). A recent RiboMeth-Seq analysis of the Arabidopsis nuc1-2 mutant indicated that 2ʹ-O-methylation at 65 rRNA sites was downregulated in this mutant (Azevedo-Favory et al., 2021). Notably, 2ʹ-O-methylation at 25S_G2396 was not downregulated in the nuc1-2 mutant (Azevedo-Favory et al., 2021). Therefore, the additive phenotype of the ncr1 nuc1-2 double mutant could result from a combination of reduced rRNA 2ʹ-O-methylation in ncr1 and abnormal pre-rRNA processing in nuc1-2.

Recent studies have linked snoRNAs to human diseases, including cancer (Abel and Rederstorff, 2019; Dsouza et al., 2021) and autoimmune syndromes (Abel and Rederstorff, 2019). Abnormal snoRNA expression is frequently detected in human breast cancers (Su et al., 2014; Dsouza et al., 2021). U3 and U8 box C/D snoRNAs, which are required for pre-rRNA processing, are upregulated in breast cancer cells, and they were shown to promote tumorigenesis (Langhendries et al., 2016). The depletion of U3 or U8 exerts an anti-tumor effect. SnoR28s and the methylation site are conserved in different organisms, including Saccharomyces cerevisiae (van Nues et al., 2011), M. musculus (Osheim et al., 2004), and H. sapiens (van Nues et al., 2011; Supplemental Figure S10), suggesting that SnoR28 might also play important roles in ribosome biogenesis and might be linked to diseases.

In summary, by elucidating the biochemical and biological functions of SnoR28s in Arabidopsis, our study advances our understanding of the mechanisms of rRNA modification and ribosome biogenesis in plant cells.

Materials and methods

Plant materials and growth conditions

All A. thaliana plants used in this study were of the Columbia-0 (Col-0) ecotype. The T-DNA insertion mutants hid2 (SALK_138192; Zhu et al., 2016) and nuc1-2 (Pontvianne et al., 2010) were described previously. The seeds were surface-sterilized, stratified at 4°C for 3–4 d, and germinated on 1/2 Murashige and Skoog (MS) medium with 1% sucrose (m/V) under a 16-h light (Philips; TLD, 36W/865)/8-h dark cycle at 22°C in a plant growth chamber. Seedlings grown in plates were harvested for further experiments or transferred to soil and grown in the greenhouse.

Generation of SnoR28.1-related deletion mutants by CRISPR/Cas9-mediated genome editing

The egg cell-targeting CRISPR/Cas9 system (Wang et al., 2015) was used to generate a set of SnoR28.1-related deletion mutants. The sgRNAs were designed using the CRISPRPLANT platform (http://www.genome.arizona.edu/crispr/CRISPRsearch.html). The locations of the sgRNAs are shown in Supplemental Figure S2A, and the sequences of sgRNAs are listed in Supplemental Data Set S4. To generate SnoR28.1-related deletion mutants (Figure 1A), nine dual spacers, each containing two sgRNAs, were separately cloned into the binary vector pHEE401E. The constructs were transformed into wild-type Col-0 plants using Agrobacterium tumefaciens GV3101 by the standard floral dip method (Clough and Bent, 1998). All deletion mutants generated by CRISPR/Cas9 were screened by PCR and confirmed by sequencing using specific primers in the T1 generation. Homozygous mutant plants lacking the Cas9 transgene by crossing were used for further experiments.

Complementation experiments

To generate the proNCR1:NCR1 (C1), proNCR1:SnoR180 (C2), proNCR1:SnoR180-SnoR28.1a (C3), proNCR1:ΔSnoR28.1c (C4), and proNCR1:ΔSnoR180 (C5) constructs (Figure 1D), a fragment containing the 2 kb region upstream of NCR1 followed by the full-length NCR1, SnoR180-only, SnoR180-SnoR28.1a, SnoR180-SnoR28.1a-SnoR28.1b, and SnoR28.1a-SnoR28.1b-SnoR28.1c sequences, respectively, were amplified from Col-0 genomic DNA by PCR and cloned into the Kpn I/Sal I sites of pCAMBIA1305 (Cambia). To generate the 35S:SnoR28.1c and 35S:SnoR28.2 constructs, SnoR28.1c and SnoR28.2 were amplified from Col-0 genomic DNA by PCR and cloned into the Pst I/Spe I sites of pCAMBIA1300 (Cambia). To generate the proNCR1:mut1-mut5 constructs (Figure 6B), mutations were introduced into the C3 construct by overlapping PCR and confirmed by sequencing. To generate the proOsSnoR28.3:OsSnoR28.3 construct, a fragment containing the 2 kb region upstream of OsSnoR28.3 followed by the full-length OsSnoR28.3 sequence was amplified from O. sativa spp. japonica cv. Nipponbare genomic DNA by PCR and cloned into the Kpn I/Sal I sites of pCAMBIA1305.

The respective constructs were transferred into A. tumefaciens strain GV3101 and transformed into ncr1 or m9 plants via the standard floral dip method (Clough and Bent, 1998). Primary transgenic plants were selected on 1/2 MS medium supplemented with 30 µg/mL hygromycin (Roche). Homozygous complemented lines were used for phenotypic analysis and RNA experiments. All primers are listed in Supplemental Data Set S4.

Histochemical GUS staining

To generate the proNCR1:GUS construct, the promoter region of NCR1 (2 kb) was amplified from Col-0 genomic DNA by PCR and cloned into the Pst I/Nco I site of pCAMBIA1305. The primers are listed in Supplemental Data Set S4. The proNCR1:GUS construct was transferred into A. tumefaciens strain GV3101 and transformed into wild-type Col-0 plants. Various tissues of proNCR1:GUS T3 transgenic plants were soaked in GUS staining buffer (10 mM EDTA [pH 8.0], 29 mM Na₂HPO₄, 21 mM NaH₂PO₄, 1 mM K₄Fe(CN)₆, 1 mM K₃Fe(CN)₆, 0.1% Triton X-100, 0.05% 5-bromo-4-chloro-3-indolyl-β-D-glucuronide [X-Gluc] and 20% methanol) and vacuum infiltrated in a vacuum chamber for 20 min. The samples were incubated overnight in GUS staining buffer at 37°C. Ethanol was used to remove the chlorophyll after GUS staining. Five independent proNCR1:GUS T3 lines, which were isolated from different T1 transgenic plants, were used for GUS staining and all of them showed similar results.

RNA extraction, RT-PCR, and RT-qPCR

Total RNA was extracted from 10-d-old seedlings using TRIzol reagent (Invitrogen). The RNA samples were treated with DNase I (Promega) to remove DNA contamination. First-strand complementary DNA was synthesized from 2 µg RNA using 5× All-In-One RT Master Mix (Applied Biological Materials Inc.). The RNA transcript levels were determined by RT-PCR or RT-qPCR. For RT-qPCR, the cDNA reaction mixture was diluted 5-fold, and 1 µL was used as a template in a 20 µL PCR system with EvaGreen 2×qPCR MasterMix-Low ROX (Applied Biological Materials Inc). ACTIN2 mRNA was detected in parallel and used for data normalization. The primers used for RT-PCR and RT-qPCR are listed in Supplemental Data Set S4.

FISH

Seedlings (10-d-old) were fixed in 4% paraformaldehyde and the nuclei were prepared as previously described (Zhu et al., 2016). The slides were postfixed in 4% paraformaldehyde for 20 min. Biotin-labeled SnoR28.1a and SnoR28.1b antisense and sense probes were transcribed using an AmpliScribe T7-Flash Transcription Kit (Lucigen). Hybridization was performed at 50°C overnight. Immunofluorescence was examined using Alexa Fluor 594-conjugated streptavidin (1:200 dilution; Life Technologies), and the samples were mounted in Vectashield mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Vector H-1200). Nuclei were analyzed under a Zeiss LSM800 confocal laser-scanning microscope at 63× magnification. About 10 individual plants were pooled as one biological replicate for nuclei purification, and 20 nuclei were observed for each sample. Three biological replicates were performed. The probes used in this study are listed in Supplemental Data Set S4.

In vitro RNA pull-down–mass spectrometry assay

Biotin-labeled RNAs were transcribed in vitro using an AmpliScribe T7-Flash Transcription Kit (Lucigen) and purified with RNA Clean & Concentrator-25 (ZYMO RESEARCH). Total protein extracts were obtained from seedling samples as described (Seo et al., 2019). Biotin-labeled RNAs (2 µg) and extracts from Col-0 were mixed in pull-down buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM DTT, 0.05% NP-40, and 1% plant protease inhibitor cocktail [Sigma]) and incubated overnight at 4°C. Prewashed streptavidin magnetic beads (New England Biolabs) were added to each binding reaction mix and incubated for 4 h at 4°C. The beads were washed 5 times using wash buffer (20 mM Tris-HCl [pH 7.0], 10 mM NaCl, 0.1% Tween-20) and boiled in SDS loading buffer, and the pulled-down proteins were separated by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue, and protein-containing gel slices were excised and digested in-gel with trypsin (1 ng/µL). The extracted peptides were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) on the Velos Pro Orbitrap Elite mass spectrometry (Thermo Scientific). MASCOT server (Matrix Science Ltd) and IPI (International Protein Index) Arabidopsis protein database were used for peptide searches.

RIP assay

RIP was performed as previously described with some modifications (Au et al., 2014). Seedlings (10-d-old) were harvested and cross-linked in PBS buffer with 1% formaldehyde. The cross-linked seedlings were ground in liquid nitrogen (N₂), the powder was suspended in Honda buffer (0.44 M sucrose, 20 mM HEPES-KOH [pH 7.4], 10 mM MgCl₂, 1.25% Ficoll, 2.5% dextran T40, 0.5% Triton X-100, 8 U/mL RiboLock [Thermo-Fisher], 1 mM PMSF, 5 mM DTT, 1% plant protease inhibitor cocktail [Sigma]), and the suspension was filtered through Miracloth. The filtered homogenate was centrifuged at 3,000 rpm for 8 min at 4°C to spin down the nuclei. Each pellet was lysed in nuclei lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS, 1 mM PMSF [Sigma], 1% plant protease inhibitor cocktail [Sigma], 160 U/mL RiboLock [Thermo-Fisher]) and sonicated 4 times on ice for 10 s at 25% amplitude with 1 min pauses. The samples were centrifuged at 4°C for 10 min at maximum speed.

For immunoprecipitation of RNA, the nuclear lysates were diluted 10-fold in IP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl [pH 8.0], 167 mM NaCl, 350 U/mL RiboLock [Thermo-Fisher]) and centrifuged for 10 min at maximum speed at 4°C to remove any remaining cellular debris. The supernatants were transferred to new tubes, GFP-Trap beads (ChromoTek) were added to the samples, and the mixtures were incubated overnight at 4°C. The samples were washed 3 times in wash buffer (150 mM NaCl, 20 mM Tris-HCl [pH 8.0], 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM PMSF, 40 U/mL RNase out [Invitrogen], 5 U/mL RQ1 DNase [Promega]). RNA-protein complexes were eluted with RNA-IP elution buffer (100 mM Tris–HCl [pH 8.0], 10 mM EDTA, 1% SDS, 40 U/mL RiboLock [Thermo-Fisher]) with constant rotation for 10 min at room temperature. The samples were centrifuged at 1,500 g for 1 min at 4°C, and the eluate was transferred into a new tube. The elution step was repeated once. To reverse cross-linking, each sample was treated with 20 µg proteinase K (Sigma) and 10 µL 5 M NaCl for 1 h at 65°C. The RNA was purified using TRIzol (Invitrogen). The primer sets used for RT-PCR analysis are listed in Supplemental Data Set S4.

Detection of 2′-O-methylation by RiboMeth-seq

To map 2′-O-methylation at specific rRNA sites in the ncr1 mutant, RiboMeth-seq was performed and the data were analyzed as previously described (Wu et al., 2021). Total RNA was extracted from the samples and hydrolyzed briefly under mild alkaline conditions. Fragmented RNAs were converted into cDNA libraries, which were sequenced to produce 150-bp paired-end reads. The reads were mapped to the reference rRNA sequences. The 5′ and 3′ ends of the RNA fragments were counted for each methylation site and combined to calculate the MethScore (Supplemental Data Set S3). The MethScore reaches a maximum of 1 when a site is fully methylated and not hydrolyzed and is lower when a site is partially methylated and has undergone some degree of hydrolysis.

RTL-P

To detect 2′-O-methylated sites in the target rRNAs, RTL-P analysis was performed as previously described (Dong et al., 2012), with minor modifications. Briefly, a mixture containing 20 ng total RNA, 1 µL (10 mM) 25S rRNA G2384-R primer (Supplemental Data Set S4), and a low (1 μM) or high (1 mM) concentration of dNTPs was denatured at 70°C for 5 min and chilled on ice. After annealing at 42°C for 10 min, 200 U of M-MLV reverse transcriptase (Promega) and 0.5 U RNasin ribonuclease inhibitor (Promega) were added to the sample. Reverse transcription was performed at 42°C for 30 min, followed by heating at 75°C for 15 min to deactivate the reverse transcriptase. The methylation levels were determined by RT-PCR. For RT-PCR, 1 µL cDNA was used as a template in a 20 µL PCR system containing 2×TaqMasterMix (CWBIO). The PCR conditions were as follows: one cycle of 95°C for 4 min followed by 20 cycles at 95°C for 30 s and at 58°C for 30 s, 72°C for 30 s. The number of PCR cycles varied depending on the template concentration. The PCR products were separated on 2.5% agarose gels. PCR signal intensities were analyzed using ImageJ software. Primer sets used for RT-PCR analysis are listed in Supplemental Data Set S4.

Primer extension to detect ribose methylation

Total RNA was used to detect 2′-O-methylation as previously described (Dong et al., 2012), with some modifications. Briefly, 8 μg DNase I-digested total RNA was mixed with 5′ FAM-labeled Gm2396-R primer (Supplemental Data Set S4) in 20 μL of AMV RT buffer (Promega) containing 1 mM or 4 μM dNTP and incubated at 65°C for 5 min. Primer extension with AMV reverse transcriptase (Promega) was performed using two aliquots of RNA in parallel in the presence of either 4 μM or 1 mM dNTPs at 42°C for 1 h. A short 25S rRNA sequence (32-nt, Supplemental Data Set S4) was prepared to precisely map the locations of the modifications. The cDNA products were separated on 10%–12% polyacrylamide-7M urea gels in parallel with sequenced 25S rDNA and analyzed using a Typhoon FLA 9,500 laser scanner.

RNA gel blotting

To detect pre-rRNA precursors, intermediates, and mature rRNAs, total RNA was separated in 1.5% (wt/vol) agarose/formaldehyde gels. The RNA was visualized and transferred via cross-linking to an Amersham Hybond N+ nylon membrane (Thermo Fisher Scientific). Electrophoresis, hybridization, and detection were performed as previously described (Zhu et al., 2016). RNA gel blot analysis was performed using digoxigenin (DIG)-labeled probes. The sequences of the probes used to detect ncRNAs are listed in Supplemental Data Set S4.

Circular RT-PCR assay

Circular RT-PCR analysis was used to detect pre-rRNA precursors as previously described (Abbasi et al., 2010; Hang et al., 2014, 2015; Zhu et al., 2016). Circular RT-PCR analysis was carried out using 5 μg total RNA. The 25S-C primers (Supplemental Data Set S4) were used for reverse transcription of circularized 25S rRNA precursors. PCR amplifications were performed with the cRT-F and cRT-R primer pairs for 30 cycles (Supplemental Data Set S4). The products were gel-purified and cloned into the pEasy-T vector (TRANSGENE), and multiple clones were sequenced using M13F and/or M13R primers.

Polysome profiling

Extracts were prepared for polyribosome profiling as described previously (Mustroph et al., 2009; Zhu et al., 2016). A total of 2,000 A260 units of the supernatant were layered onto a linear 15%–60% sucrose gradient poured with a peristaltic pump (LEAD FLUID, BT101S). Following ultracentrifugation in a Beckman L8-M Ultracentrifuge with an SW55 rotor at 237,000× g for 1.5 h at 4°C, the gradients were analyzed using a 185 Gradient Fractionator (ISCO Lincoln, NE) attached to an ISCO UA-5 UV detector for continuous measurement of the absorbance at 254 nm. All fractions were collected using a fraction collector, and several selected fractions were subjected to RT-PCR analysis. The primer sets used for RT-PCR analysis are listed in Supplemental Data Set S4.

ChIRP and ChIRP-qPCR analyses

ChIRP was performed as previously described, with some modifications (Chu et al., 2012; Zhu et al., 2016). Antisense DNA probes were designed against the full-length SnoR28.1a sequence and biotinylated at the 3′ end (Invitrogen). A set of probes against SnoR126 RNA was used as the control. Wild-type seedlings (1 g) were cross-linked in 1% (vol/vol) formaldehyde (Sigma) at room temperature for 20 min under a vacuum. Cross-linking was quenched by adding 0.125 M glycine and incubating for 5 min. Nuclei were purified in Honda buffer as aforementioned in RIP experiment. The purified nuclei were washed with transcription buffer (125 mM Tris-HCl [pH 7.5], 500 mM KCl, 25 mM MgCl₂, 10 mM DTT, 500 mM sucrose, 25% [vol/vol] glycerol), and sonicated using a Bioruptor ultrasonicator (Diagenode). The chromatin was diluted in 2 volumes of hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl [pH 7.0], 1 mM EDTA, 15% formamide, 0.1 mM PMSF, protease inhibitor, and RNase inhibitor). After preclearance using Streptavidin Sepharose beads (GE Healthcare), 100 pmol of probes were added and mixed by end-to-end rotation at 37°C for 4 h. Following the addition of prewashed/blocked Streptavidin Sepharose beads (20 μL), and the sample was rotated at 37°C for 30 min. The beads were washed 3 times with high-salt wash buffer (2× SSC, 0.5% SDS, 1 mM DTT, and 1 mM PMSF) and 3 times with low-salt wash buffer (0.1× SSC, 0.5% SDS, 1 mM DTT, and 1 mM PMSF) for 5 min each at 37°C. DNA and RNA were purified and analyzed by qPCR. Probes and primer sequences are provided in Supplemental Data Set S4.

Statistical analysis

For all multiple comparisons, the significance of the difference between different groups was analyzed by one-way analysis of variance (ANOVA) along with Bonferroni’s multiple comparison test at significance levels of 0.05 and 0.01 using SPSS statistics. Asterisks indicate significant differences between the wild type and the indicated mutants (P < 0.05). Different lowercase letters above the bars indicate significantly different groups (P < 0.01, Supplemental Data Set S5).

Accession numbers

Sequence data for the genes described in this study can be found in the TAIR database (https://www.arabidopsis.org) and NCBI under the following accession numbers: SnoR28.1a (AT5G02675), SnoR28.1b (AT5G02665), SnoR28.1c (AT5G02655), SnoR28.2a (AT5G05385), SnoR28.2b (AT5G05375), SnoR126 (AT5G09495), HID2 (AT1G06233), OTC (AT1G75330), ACTIN2 (AT3G18780), UBC21 (AT5G25760); OsSnoR28.3a (AJ307917), OsSnoR28.3b (AJ307918), OsSnoR28.3c (AJ307919), and OsSnoR28.3d (AJ307920).

The RiboMeth-seq raw data in this study have been deposited into the National Genomics Data Center (bigd.big.ac.cn) under accession number PRJCA003515 (RiboMeth-seq of WT under accession code CRA003263; RiboMeth-seq of ncr1_rep1 under accession code CRA005548; RiboMeth-seq of ncr1_rep2, mut4/ncr1, and mut5/ncr1 under accession code CRA007577).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Pleiotropic developmental defects of SnoR28.1 mutants.

Supplemental Figure S2. SnoR28.1s are functionally redundant in plant development and growth.

Supplemental Figure S3. Sequence alignment of Arabidopsis SnoR28 and complementation of ncr1 by SnoR28.2a.

Supplemental Figure S4. Identification of SnoR28.1-associated proteins.

Supplemental Figure S5. Mapping of the 3′ and 5′ extremities of pre-rRNA by circular RT-PCR with cDNA reverse transcribed with the 25S-c primer.

Supplemental Figure S6. RT-PCR analysis of the expression of the VAR1-VAR4 45S rRNA variants in WT, ncr1, and C1/ncr1 plants using the p5 and p6 primers.

Supplemental Figure S7. SnoR28.1s are not associated with rDNA.

Supplemental Figure S8. Analysis of the functional conservation of SnoR28.

Supplemental Figure S9. Genetic interaction of ncr1 with nuc1.

Supplemental Figure S10. Extra conserved snoRNA sequences are complementary to adjacent rRNA target sites.

Supplemental Data Set S1. List of candidate ncRNA genes.

Supplemental Data Set S2. Identification of SnoR28.1-associated proteins by RIP-MS.

Supplemental Data Set S3. MethScores of WT and ncr1 plants.

Supplemental Data Set S4. Primers and probes used in this study.

Supplemental Data Set S5. Tables for statistical analysis.

 

Y.C., K.Y., and W.Q. designed the research. Y.C., J.W., X.Y., J.S., X.D., and Y.L. performed the experiments. Y.C., S.W., L.S., D.Z., K.Y., and W.Q. analyzed the data. Y.C., X.W.D., and W.Q. wrote the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) is: Weiqiang Qian (wqqian@pku.edu.cn).